Sensing of Different Human Telomeric G-Quadruplex DNA Topologies

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Sensing of Different Human Telomeric G-Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques Paulami Mandal, Dibakar Sahoo, Saumen Saha, and Joydeep Chowdhury J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07856 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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The Journal of Physical Chemistry

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Sensing of Different Human Telomeric G-Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques Paulami Mandal*a, Dibakar Sahoob, Saumen Sahaa, Joydeep Chowdhurya a

Department of Physics, Jadavpur University, 188, Raja S. C. Mallick Road, Kolkata, West Bengal 700032,India.

b

*

School of Physics, Sambalpur University, Odisha 768 019, India.

Corresponding author.

E-mail address: [email protected] (P. Mandal)

ACS Paragon Plus Environment

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Abstract Current article describes how does a natural alkaloid Allocryptopine (ALL) able to differentiate two forms of biologically relevant human telomeric (htel22) G-quadruplex DNAs (GQ-DNA) depending upon the presence of K+ and Na+ ions by steady state and time-resolved spectroscopic techniques. For both interactions predominant involvements of static type quenching mechanism with the negligible influence of dynamic collision are established by UV-Vis absorption and fluorescence emission study which is further supported by fluorescence lifetime measurements. ALL exhibits appreciable affinity towards both GQ-DNAs. Both the mixed-hybrid (3+1) quadruplex structures in K+ ions, and basket-type anti-parallel quadruplex structure in Na+ condition are converted to parallel types in the presence of ALL. FID experiment revealed modest selectivity of ALL to the both quadruplexes over duplex DNA along with higher selectivity for anti-parallel types among the two quadruplexes via groove and/or loop binding, which is distinct from the conventional -staking of the ligands on external G-quartets. ALL stabilized both GQ-DNA topologies moderately. The differences in the dynamics of ALL within both DNA environments have been demonstrated vividly by the time-resolved anisotropy measurements using wobbling-in-cone model. These results suggest groove binding with antiparallel G-quartet with high affinity and moderate loop binding with mixed hybrid G-quartet accompanied by the partial end-staking additionally in both the cases. Our conclusions are further supported by steady state anisotropy measurements and molecular docking. Present investigation could be used in the development of biocompatible anti tumour/anti cancer agent targeting particular GQ-DNA conformation.


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I. Introduction. Since last two decades a large range of new families of anticancer agents with novel action mechanisms have been introduced but still the drugs which are able to function by interplaying with DNA is one of the significant research areas in our modern world. It is wellknown that DNA can exist in diverse structural forms like parallel-stranded (ps) DNA, single stranded hairpins, triplexes, i-motif, and tetraplexes or quadruplexes apart from classical double helix and that has evoked new molecular targets for novel families of anticancer agents1. However, among these structures only recently G-quadruplex structure is found to be biologically crucial. G-rich sequences are present in the telomeric DNA repeats and also at the promoters of numerous oncogenes /proto-oncogenes which assumes non-canonical four-stranded forms recognized as G-quadruplexes

2,3

. These structures are stacks of four guanine bases

arranged in a square planar configuration and balanced by Hoogsteen hydrogen bonding 4. Also, G-quadruplexes persist in different topologies either parallel or antiparallel, or in both conformations (termed as mixed-hybrid) depending upon the DNA-strand orientation or loops in the presence of extrinsic monovalent cations like Na+ and K+

2,3

. Different G-quadruplex

topologies are shown in Figure 1a. In Na+ solution, the four-repeat human telomeric sequence d[AGGG(TTAGGG)3] adopts an antiparallel stranded basket-type G-quadruplex

5,6

. Whereas in

K+ solution, same sequence forms multiple predominantly intramolecular (3 + 1) mixed-hybrid G-quadruplex configurations

3,7-8

. Nevertheless, till date not much is known about in vivo

quadruplexes. However amongst many activities, one of the important biological activities of the G-quadruplex structures is to inhibit the activity of telomerase enzyme. This enzyme is responsible for the eternal life period of cancer cells and is effective in approximately 85% of tumours. Thus any small ligands which can facilitate and stabilize quadruplex generation could be treated as probable telomerase inhibitors and consequently an anti- tumour or anti-cancer



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drug9. Apart from potential use in therapy, these quadruplex binders are also important in the development of various DNA nanomachines within, such as, molecular diagnostics, biosensing and imaging techniques. In all of the above mentioned applications, fluorescence is used as an effective sensing technique involving photostable and chemically stable fluorophores with high fluorescence quantum yields

10,11

. In imaging, turn‐on fluorescent probes are popular although

fluorescence quenchers also have been proved to be very much useful. Nonetheless, nucleic acids are fascinating construction pieces for guided assemblage of nanostructures12. From a drug discovery perspective, designing new profoundly selective molecules which can target and stabilize or disrupt G-quadruplex configurations among the most abundant B form duplex DNA is immensely desirable. Generally small molecules bind with duplex DNA either by covalent binding (alkylation/inter and intra strand cross linking ) or by non-covalent binding (electrostatic/surface binding with the anionic phosphate backbone, groove binding via hydrogen bonding / by van-der Waals interaction and intercalation within the two bases). However, quadruplex and duplex structures of DNA differs completely from each other for having contrasting geometries and functional groups13. Consequently, small molecules usually interact with the G-quartets by - staking interaction, or interact with the grooves/loops/ negative phosphate backbones of GQ-DNAs14,15. But still, in targeting G-quadruplexes the challenge remains in designing ligands which can stabilize quadruplexes with high selectivity and specificity over diverse DNA forms16-19. However, till date a number of small molecules have been discovered which can bind and induce telomeric GQ-DNAs, for instance, anthraquinone derivatives

20

, porphyrins

21

, acridines

22

,

perylenes 23, triazines 24, aminoglycosides 25, benzimidazole derivatives 26, and natural elements like ascididemin28, telomestatin27, alkaloids and their derivatives 29,30 are recognized as probable


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anticancer agents. Nowadays, among these, natural elements are coming up as a promising candidate in the development of novel drugs with antitumor activity for having salient features like large variability in their chemical structures, huge availability from natural sources, cheap extraction processes, and most importantly, in general low toxicity. Present investigation involves naturally occurring isoquinoline alkaloid (IQA) Allocryptopine (ALL) (Fig.1b) as a small molecule which is mainly isolated from the plant families Berberidaceae, Papaveraceae, Fumariaceae, and Rutaceae. ALL is the component of numerous phyto preparations. As other IQAs, it also helps the plants in combating the biotic stress31. Moreover, IQAs possess a wide spectrum of biological activities e.g., anti-thrombotic, anti-spasmodic, anti-inflammatory, antifungal, neuro-protective, anti-bacterial, anti-parasitic, and anti-viral activities apart from their multiple actions on the cardiovascular system. Importantly, ALL has anti-tumor activity, which could be exploited successfully in anti-cancer therapy33. Current article aims to describe the critical differences in effects of the alkaloid ALL upon the optical, structural and dynamical aspects of the two different topologies of G-quadruplex DNAs in two K+ and Na+ ionic conditions using several steady state and time-resolved spectroscopic tools and molecular docking. Present investigation could be further employed as a model to other ligand-G-quadruplex DNA interactions. Herein, we have estimated whether the natural alkaloid ALL could able to distinguish antiparallel topology (under Na+ conditions) and mixedhybrid topology (under K+ conditions) of htel22 quadruplex DNA and its selectivity towards quadruplexes over duplex DNAs. Altogether, this potential study helps us to differentiate the characteristics and actions of two important GQ-DNA topologies (anti-parallel and mixed hybrid) based on their configurational dissimilarities, and also supports us to understand



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activities of the antitumor and antivirus agents which would be very much useful in developing novel and highly effective gene directed therapeutics. II. Maerials and Methods Materials. Our study involves 22-mer human telomeric (htel22) DNA oligonucleotides (5'AGGGTTAGGGTTAGGGTTAGGG-3') of HPLC grade were bought from Eurofins Genomics India Pvt. Ltd without further purification. Alkaloid Allocryptopine (ALL) (98% pure) was supplied from Aldrich and was used in our study after checking the purity of the alkaloid through impurity emission testing in the desired wavelength region. We have taken fresh TE buffer. Oligonucleotide solutions are prepared by dissolving it in 10mM Tris-HCL, 1mM EDTA, 100mM KCL and NaCl at pH 7.4 separately 34. At first annealing of htel22 was done at 900C for 10 min and then stored at 40C for 48hrs 35. The concentration of the GQ-DNA stock solution was 100µM. Formation of (3+1) hybrid GQ-DNA topology under K+ ions and antiparallel stranded basket type GQ-DNA topology in Na+ condition have been confirmed through CD before each experiment. Absorption and Fluorescence Emission Study. At 296 K (ambient temperature), we have taken the steady state absorption spectra using an absorption spectrophotometer Shimadzu UVvis 2401PC and also have recorded fluorescence emission spectra of diluted sample solutions through F-7000 fluorescence spectrophotometer(Hitachi). We have used rectangular quartz cells of 1 cm path length in both the cases. Circular Dichroism Study. We have used CD Spectrometer, modelJ-815-150S, connected with a thermostable cell holder to record our CD spectra. Here we have used quartz cuvette of 0.2cm path length at 200C, TE Buffer and 100mM of the both salts. Here we obtained our spectra as a function of wavelength (200–350 nm) which is the averages of three scans at a scan speed of


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100nm/min with baseline correction from the blank buffer. Equilibration has been ensured by recording each spectrum after 10 minutes of the addition of both DNAs. Fluorescence Intercalator Displacement Assay.

Fluorescence Intercalator Displacement

titrations were performed at room temperature in buffers. The DNA solutions were prepared by mixing of 0.5µM Thiazole Orange (TO) with 0.25µM of both GQ-DNAs. An increasing amount of ALL was added successively by a 5 mins of equilibration period before the fluorescence spectrum (excitation at 501nm and emission at 510-700nm) was recorded. CD melting Study. DNA melting study in the absence and presence of the alkaloid was performed through JASCOJ-815-150SCD Spectrometer at the heating rate of 10C/min. All CD melting experiments were monitored at 295nm and the temperature was increased from 200C to 900C. The CD melting experiments were conducted in TE Buffer with100 mM of both salts at pH 7.4. The Tm has been calculated by taking the 1st derivative of the melting profiles. Samples were tightly capped by Teflon stoppers36. Studies of Steady State and Time resolved Anisotropy. Steady state anisotropy (r) was assessed using Varian Cary Eclipse (USA) spectro-fluorimeter. In our study anisotropies were calculated by using the relation 37:

r= Where, IVV and IVH are the intensities of parallel and perpendicular polarized emission with vertically polarized excitation, and IHH and IHV are the intensities of horizontally and vertically polarized emission while excited with horizontally polarized light. IHV/IVV describes instrumental



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correction factor G. This correction is contrived due to any alteration in the emission channel sensitivity for the vertically and horizontally polarized components. The time-resolved fluorescence decays and polarized emission decays are measured by the single-photon-counting technique. The time-dependent fluorescence anisotropy function r(t) is demonstrated below:37 r(t) =

( )

( )

( )

( )

Here, IVV(t) and IVH(t) describes the polarized fluorescence decays. Here we have excited the samples at 340 nm by a picosecond diode (IBH Nanoled -07). Corresponding emission was recorded at a magic angle polarization using a Hamamatsu MCP photomultiplier (2809U). The time correlated single-photon counting (TCSPC) set up has an ortec 9327 CFD and a Tennelec TC 863 TAC. Collection of the data is made by a PCA3 card (Oxford) as a multichannel analyzer. 25 ps is the FWHM of the system response. Width of the channel is 12 ps/channel. Deconvolution of fluorescence decays has been made by IBH DAS6 software. Docking Studies. The ligand ALL molecule was optimized using density functional level of theory (DFT). In the DFT calculations, B3LYP functional inclusive of 6-31++G (d,p) basis set had been adopted. The 6-31++G (d,p) is a split valence basis set with polarization functions “d” and “p”. The polarization functions allow the electron distribution to remain polarized in the computational run towards estimation of the optimized molecular structure of ALL. ALL contains Nitrogen and Oxygen atoms, both having lone pair electrons. Lone pair electrons on these atoms are loosely bound and they settle at larger distances from the corresponding nuclei in comparison to bonding or core electrons. DFT calculations take into account the effect of lone pairs to estimate the optimized geometry of the molecule. Gradient correlated Becke’s three


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parameter hybrid exchange (B3) and Lee-Yang-Parr correlation (B3LYP) functional has been used in the DFT calculation

38,39

. However, the vibrational frequency estimations do not exhibit

any imaginary value, signifying the attainment of local minima on the potential energy surface of the ligand molecule. The optimized molecular structure of ALL is shown in Figure S1. DFT calculations were accomplished by software Gaussian 03 suit in the Windows operating system. The optimized structure of the ligand ALL was docked one by one with antiparallel basket type and (3+1) hybrid kind GQ- DNA with the aid of Autodock 4.2 software. The crystal structures of antiparallel basket type and mixed hybrid GQ-DNA were obtained from the Protein Data Bank (PDB)40,41 with the PDB identifier 143D(antiparallel GQ-DNA) and 2HY9(mixed-hybrid GQDNA) respectively. Docking calculations were accomplished using Atodock 4.2 software. It deploys an empirical scoring function on the basis of free energy binding

42

. Autodock

Lamarckian Genetic Algorithm (LGA) was used to estimate the binding stance of the ligand molecule.

III. Results & Discussion: Steady State UV-Vis absorption spectra. In order to reveal ground state interaction in between the natural alkaloid ALL and two forms of GQ-DNAs in the presence of K+ and Na+ ions respectively, UV-Vis absorption technique was used. This was employed to monitor the modifications in absorption spectra of ALL as we have enhanced the concentrations of two GQDNAs gradually (up to 8 µM)(Fig.2). Concentration of ALL was held fixed at ~ 100 µM all over the UV-vis experiment. ALL absorption spectra are found to consist of a wide band with a maximal at 283 nm. The stepwise addition of the two GQ-DNAs to the alkaloid solution contributed a hyperchromic effect to the 283 nm absorption band of ALL. In addition, there is a blue shift of 5nm in K+ and 3nm in Na+ at this 283nm peak position. These significant



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modifications in the ALL absorption spectra indicate occurrence of a noticeable change in the electronic environment of ALL as it binds with the both GQ-DNAs43. Moreover, the blue shifts in both spectra suggest that the polarity around ALL molecules alters from lower to higher polarity due to its interaction with two DNAs. Absence of the isosbestic point in the absorption spectra implies that ALL binds with the both GQ-DNAs probably via more than single type of mode 44. Thus outcome of our absorption study surely indicates an influence of ALL on the two types of GQ-DNAs in their ground states. Fluorescence Emission Studies.

To add valuable information regarding the structure and

dynamics of both GQ-DNA topologies we had measured the intrinsic fluorescence changes of the alkaloid ALL in the presence of both htel22 GQ-DNA structures in the K+ and Na+ ions. In our study, ALL had been excited at 285nm in the presence of GQ-DNAs (Fig.3). It should be noted that the absorbance of both GQ-DNAs at 285nm was very negligible. From the Fig.3a &3b it is apparent that with the increasing concentrations of GQ-DNA from 0 to 8 µm in the both K+ and Na+ ionic conditions the fluorescence emission spectra of the alkaloid had been quenched gradually. Throughout the experiment concentration of ALL was kept constant at 50 µM. However it is logical to presume that at the high concentrations of DNAs inner-filter effect may occur during the quenching of ALL fluorescence. Therefore, corrections of the steady state fluorescence intensity for the inner-filter effect were accomplished by the given equation37:

= where ODqex and

(

2

+

2

)

are the absorbance and emission intensity of DNAs at the excitation

wavelength 285 nm. However corrections were found to be negligible. Gradual mixing of the two DNAs (Fig.3a&3b) caused a quenching in the emission profile of ALL (~ 49% in K+ and


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71% in Na+ at 8 µM of each GQ-DNA). Although, the fluorescence quenching of ALL in the presence of two GQ-DNAs indicates an appreciable binding interaction but in the presence of Na+ ions the binding interaction is more pronounced than in the presence of the other ion. For both the cases to find the binding modes of ALL with GQ-DNAs we have employed SternVolmer equation37: = 1+

[ ]=

 

Here F0 and F define the steady state fluorescence emission intensity in the absence and presence of quencher, respectively. KSV (=kq0) is the SV quenching constant and [Q] is the concentration of DNA. kq is the bimolecular quenching constant and 0 is the unquenched lifetime of the molecule which is 2.445 ns for ALL. KSV was estimated in both the cases to assess the efficiency of fluorescence quenching of ALL by DNAs. The linearity of SV plots hint the involvement of only one type of quenching process, i.e. either dynamic or static kind. Contrastingly, upwardly curved SV plot reveal the existence of both static and dynamic quenching processes45. Present results show that for the two different ion conditions corresponding SV plots (insets of Fig.3a &3b) are almost linear in nature which indicates predominant involvement of either static or dynamic quenching processes in the both cases. The values of KSV and kq are enlisted in the Table1 and the kq for both the cases are found to be much higher compared to the maximum scatter collision quenching constant (2x1010 L.mol-1.s-1) for various quenchers with bimolecules 45

. This confirms that in the two interactions there prevails mostly static kind of quenching. Thus

SV results estimates that ground state strong complex formation is the key cause behind the appreciable fluorescence quenching while dynamic collision might have some negligible influence46,47. However, these values reveal that apart from dynamic contributions, ground state



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complex formation in between the alkaloid ALL and GQ-DNA topology in the Na+ ion conditions is much stronger and more stable than that in the K+ ion condition. Moreover, the KSV and kq values for both GQ-DNAs lead us to speculate a few probable reasons behind this fluorescence emission quenching of ALL, such as: (i) intercalation of ALL in between GQ-DNA quartet or (ii) end staking at the top or bottom end or/and (iii) binding in the external loops and/or grooves of GQ-DNA 25,35. But energetically the unfolding of an intercalation site, buried in the stack of G-quartets is very low. Nonetheless, there is no superposition of the emission of ALL and absorption spectra of any nucleobases, which makes us to cancel out the probability of the resonance energy transfer (FRET). Therefore, primarily our steady state measurements attributes to the prospect of exterior loop and/or groove binding accompanied by the partial end staking of ALL to the both G-quadruplexes in the presence of two ions 48. Time-resolved Measurements. To have the deep insight about the binding mechanism we have also performed different time-resolved measurements upon the ligand-quadruplex interactions. From the time-resolved lifetime measurements it is found that the lifetime decay profile of alkaloid follows a double exponential fitting (Fig.S2). In our study we have used the average lifetime of ALL of these two lifetime components which is found to decrease gradually with the increasing concentrations of DNAs both in the presence of K+ and Na+ ions (Table S1) which is indicative of some sort of involvement of dynamic quenching effect in both the cases. In order to gain more information about the lifetime data we have calculated the dynamic quenching constant of these interactions by using the Stern-Volmer Equation37:  

= 1+ KD [Q]


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where 0 and  are lifetime of ALL in the absence and presence of two types of GQ-DNA, KD and[Q] are the dynamic quenching constant and the quencher’s concentrations respectively. By employing this SV equation we have plotted the lifetime data of ALL at different concentrations of GQ-DNA (inset of Fig.S2) for both ionic conditions. The plots are linear in nature and the corresponding dynamic quenching constants are calculated to be 3.25 x 104 in the presence of K+ and 1.03 x 105 in Na+ condition. The corresponding bimolecular quenching constants are found to be much higher than the theoretically estimated value generally arising from the diffusion properties49. These results surely reveal the assured presence of some other additional quenching mechanism other than static type quenching in our study. Hence, lifetime results further corroborate our previous predictions about the dominating presence of static quenching process along with insignificant contribution of dynamic quenching processes in the two interactions. Estimation of Binding Parameters. Next to further assess the binding constant (Kb) and the corresponding stoichiometry (i.e. n: number of binding sites) for the interactions of ALL with two GQ-DNAs in the presence of two ions K+ and Na+, fluorescence titration data have been employed in the following equation 43: (

− )

=

+

[

]

where F0 and F are the fluorescence intensities of the ALL without and with the two GQ-DNAs. Concentrations of two G-quadruplexes are denoted by [DNA]. Kb and n have their usual meaning.



14

It could be observed from the Fig.3c that the log(F0-F)/F vs log[DNA] plot is linear in nature. Corresponding Kb and n values are presented in the Table 2. The value of n for the alkaloid-DNA interaction under the K+ condition reflects the presence of single binding site and less stable complex formation while that in the presence of Na+ signifies involvement of more than one binding sites and stronger as well as more stable complexation in comparison to the preceding one. The values of Kb for the ALL-GQ-DNA interactions suggest that in the K+ ionic condition there was a modest ligand-quadruplex interaction but in the presence of Na+ the interaction was much stronger than the previous one50. Thus, much more binding affinity of ALL towards GQDNA species in Na+ than that to the GQ-DNA structures in the K+ ion conditions is revealed from the Kb values. The reasons behind the affinity of our alkaloid towards both GQ-DNAs might be attributed to the ten-member heterocyclic ring of ALL, consisting of one tertiary nitrogen and carbonyl group, stabilized by strong electrostatic interaction, which helps the alkaloid to bind with the bases of both DNAs via hydrogen bonding, van-der-wal interaction and π-π staking and probably by electrostatic interaction with the phosphate backbone as well. Differences in binding constants and binding sites demonstrate the specificity and selectivity of the ligand towards the structural differences of both GQ-DNAs (antiparallel and mixed hybrid) under the two different ion conditions. Circular Dichroism (CD) Spectroscopic Studies. In order to get further understanding to the differences in the ALL and DNA interactions in the presence of two different ions we have employed CD spectroscopy. Here in our study, CD spectroscopy discloses the changes occurred in the different GQ-DNA topologies, the effects of metal ions and stability of GQ-DNAs due to the interaction with ALL in the far-UV region (190-350 nm). Usually an anti-parallel quadruplex structure is represented by the positive peaks at nearly 295nm and 240nm, accompanied by a


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negative band at 260 nm. Whereas a negative peak close to 240nm and a positive peak around 260nm signify a parallel topology of quadruplex DNA. Mixed-hybrid structure is characterized by two positive maxima at 290nm and 270nm. Fig. 4a demonstrates the CD spectrum of the oligo htel22 under the K+ condition where the DNA concentration was fixed at10 µM. Here we obtain a broad positive band at around 290 nm with a tiny positive peak at 273 nm along a negative band around 234 nm - 240nm region, which possibly suggests formation of mixedhybrid (3+1) quadruplex configuration

35,41

. There is also a hump at around 250 nm which

seemingly indicates the presence of meager portion of unvaried single – strand GQ-DNA51. As we add ALL regularly to the DNA solution, the 234 nm trough along with a hump at 270 nm become more salient accompanied by a decline at 290 nm peak. Moreover, the 250nm band gets diminished gradually. Nonetheless, as peak at 270 nm and a dip at 240nm commonly attributes to parallel GQ-DNA52, here we assume that a larger portion of parallel quadruplexes were created due to the interaction of ALL with hybrid GQ-DNA. Hence in conclusion, we presume that under K+ ions when ALL is absent, mixed (3+1) quadruplex topologies were formed along with a tiny portion of unvaried single – strand GQ-DNA. These single-strand GQ-DNAs were further converted to parallel ones upon the addition of ALL (Fig.4a). Most likely, under K+ condition, GQ-DNA was transformed from mixed to parallel topology by ALL and at the same time further parallel GQ-DNA generation was facilitated. Contrastingly, the CD spectrum of the htel22 G-quadruplex under the Na+ ions (Fig.4b) consist of a 263nm negative band along with 294 nm and 240 nm positive bands which confirm the formation of the basket – type antiparallel structure of the free GQ-DNA in this ion condition43,53. Addition of ALL, lead to the gradual decrement of 240nm, 263nm and 294 nm bands with a slight red shift of 1 nm in the 294 peak. Also the intensity of the band around



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280nm got increased slightly. These modifications in CD spectra suggests complexation of ALL with GQ-DNA in Na

+

condition which further induced a change in GQ-DNA from the

antiparallel configuration to parallel one keeping its global secondary structure unaltered 53. CD Melting Studies. Thermal stability of the quadruplexes in the presence of ligands was assessed from the CD spectroscopy by comparing the melting temperatures (Tm: midpoint of the melting transition is the melting temperature) of DNAs at 295nm wavelength. In our case, melting investigations were carried out for htel22 quadruplexes in K+(mixed-hybrid topology) and Na+ (antiparallel topology) ions respectively by following the changes in ellipticity at 295nm(Fig.5). In the absence of the ligand ALL, the Tm of the GQ-DNA in the K+ and Na+ conditions are 59.80C and 540C respectively. In the presence of ALL (Conc.~ 10 µM) the melting temperature of GQ-DNA in the presence of K+ increased by 3.60 while under Na+ condition it was enhanced by 30C indicating a moderate stabilization of the both GQ-DNAs upon binding with ALL 5. Therefore, thermal melting results refer to the almost similar propensity of ALL to form stable interactions with both GQ-DNA topologies by enhancing the stability of the both GQ-DNAs with respect to the native GQ-DNAs. Fluorescence Intercalator Displacement Assay Experiment. Next to have deep insight to the nature of binding of ALL to both GQ-DNA species in the presence of K+ and Na+ ions and at the same time to evaluate the affinity and selectivity of ALL to the GQ-DNAs above the duplex DNA, an elementary and rapid technique, fluorescence intercalator displacement assay (FID)53 had been exploited here. It is the fall of fluorescence of Thiazole Orange (TO) due to competitive displacement of DNA by the putative ligand. DC50 estimates the affinity of a molecule for a particular DNA topology. This is actually the amount of ligand needed to produce a 50% fluorescence reduction which is reflected by a 50% displacement of the fluorescence probe.


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Here, we have analyzed the binding capacities of ALL with two different topologies of quadruplex DNA. Fig.6a displays the fluorescence emission spectra of TO – GQ-DNA mixtures without and with ALL showing quenching of emission intensity of the mixture due to gradual titration of ALL into TO-GQ-DNA solutions. Our investigation revealed that 2.5µM ALL failed to dislocate TO entirely from TO-GQ-DNA complexes. This ligand concentration is conventionally employed in these types of assay studies. In our case, only 37% TO

was

dislocated from TO bound GQ-DNA under K+ condition by 16 µM of ALL while only 26.5% of TO from TO-GQ-DNA complex under Na+ condition was displaced at 11.8 µM of ALL. Thus here determination of

GQ

DC50 was not possible for both the cases. Moreover, 15µM ALL

displaced only 46% TO from TO-duplex DNA complex (Fig.6b). Hence the selectivity could not be evaluated conventionally by the dsDC50/GQDC50 as it would implicate unreliable extrapolation of the plot. Alternatively, we have introduced here estimated selectivity which is defined in the following way 54: First dsDC2.5µM value for the duplex DNA-TO complex has been calculated and was found to be 10% (Fig. 6b), then the necessary ALL concentration (GQC) to attain equal amount of (10%) dislocation of TO from TO-GQ-DNA under K+ and Na+ conditions were measured and it was found

GQ

CK =1.9µM and

GQ

CNa= 0.8µM respectively, at the end, we have

defined the estimated selectivity as 2.5/GQC and here in this study this was computed to be 3 in the Na+ ions and 1.3 in K+ conditions (Table S2). These values hint the modest selectivity of ALL towards both quadruplex structures over duplex DNA. However, ALL specifically shows more selectivity to the anti-parallel topology of GQ-DNA in the presence of Na+ than the mixedhybrid structures in the presence of K+ over ds-DNA. Thus from our FID experiment it could be demonstrated that ALL fastens to the both GQ-DNAs with reduced affinity than TO, and/or ALL probably ties to GQ-DNAs in a mode different from



18

TO. Our fluorescence quenching results have already indicated moderate affinity of ALL towards mixed-hybrid GQ- DNA in K+ and strong affinity to the antiparallel basket type GQDNA in Na+. Henceforth, this fractional dislocation of TO from the GQ-DNA-TO composite firmly implies presence of a indirect fight in binding of ALL to a site like quadruplex grooves and/or loops. This binding mode of ALL is clearly distinct from TO which typically binds to GQ-DNA via -staking on its external quartets54. Although both mixed-hybrid (3+1) and antiparallel basket quadruplexes of 22 mer human telomeric DNA have accessible quartets at the exterior which can easily welcome planar binders but ALL might not adopt a stretched configuration favouring a non typical binding mode (Scheme 1)in our case. Thus our FID results suggest that ALL shows higher selectivity towards the telomeric quadruplex form in Na+ than that in the presence of K+. Steady state anisotropy. Anisotropy measurement allows us to estimate the extent of rigidity offered by surrounding environment

37

. The change in the steady state anisotropy of ALL with

the change of concentrations of the two GQ-DNAs is presented in the Figure 7a. We obtained here a sharp rise in the steady state anisotropies with the subsequent mixing of two types of quadruplexes, which indicate that ALL were rotationally far more restricted inside the both DNA environments compared to its free state. In the presence of 7 µM of GQ-DNA, under K+ condition the increment in the rotational restriction of ALL is almost 2 times whereas in Na+ condition it is 6 times than its free state. Time resolved Anisotropy. In order to be more aware of the rotational dynamics of ALL inside both G-quadruplexes we have carried out time-resolved anisotropy decay analysis of ALL in the buffer solution. Fig.7b presents time resolved anisotropy decay curves and the corresponding data are compiled in the Table 3& 4. Without G-quadruplexes, we obtained single exponential


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anisotropy decay of ALL with rotational relaxation time 608 ps because the buffer provides an uniform surrounding for ALL. In Contrast, mixing of two GQ-DNAs to ALL delivered biexponential decay profiles of ALL with the two distinct rotational relaxation times arising out of free and constrained ALL respectively. For both the quadruplexes, when ALL is constrained to DNA a slow component emerges and this is the rotational time appearing from the DNA constrained ALL. Moreover, there is a fast component signifying the rotational time of the unbound alkaloid. Corresponding slow and fast components of rotational relaxation times for both the cases are enlisted in the Table3.The hydrodynamic volumes of unbound ALL and DNA fastened ALL could be derived from the rotational relaxation times (r) through the StokesEinstein’s relation as given below35: r =

=



where Dr, and V are the rotational diffusion coefficient, viscosity of the medium, and hydrodynamic volume of the complex at absolute temperature T respectively.

The

hydrodynamic radius of the ALL is found to be 9.1Å whereas after binding with GQ-DNA in K+ and Na+ it is estimated to be 17.94 Å and 13.22 Å respectively (Table 4). These increments in the hydrodynamic radius of our alkaloid ALL in the presence of both DNAs further corroborate our presumption of the formation of association complex between ALL and the two GQ-DNA topologies. Additionally, wobbling motion of ALL inside the two quadruplexes has also been investigated employing wobbling-in-cone model. Conferring to the present model the rotational anisotropy decay function is given below44,55: r(t)= r0[ exp

(



)

(

+ (1 − )exp (

)

)]



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20

where S2 =  is the order parameter defining the degree of restriction on the wobbling in cone orientational motion,  = pre-exponent factor of the slow part of rotational relaxation time, r0 = limiting anisotropy. S2 satisfies the inequality 0 S21, where S2=0 denotes unrestricted reorientation and S2=1 when no wobbling in cone orientational motion occurs. Present model demonstrates that the faster rotational relaxation (fast) is the motion of a constrained rotor or probe which has its transition dipole moment (µ) enduring a orientational diffusion in a semicone of angle θ about a hypothetical axis. Hence faster and slower rotational lifetime constants could be described as follows:



−

=

here  is the decay time constant of the wobbling motion of the probe. The semicone angle θ is obtained from the order parameter in the following way: S2 = [ (cos θ)(1+cos θ)]2 The wobbling-in-cone diffusion constant D when θ 300 can be derived from the relations given below: D





where θ is in radians. Nonetheless, when θ 30o D={(1-S2)}-1

(

)

+

+

(6 + 8 −


− 12

−7

)

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where x= cos θ In the Table 4 the calculated values for Dr, r, , D and S are provided. Upon binding with quadruplexes, ALL experiences a reorientation because of constrained internal rotational motion above the overall rotation. As discussed earlier, S refers to the spatial restriction of dynamics of the fluorescence probe in the restricted space. However, free ALL in buffer possess fast unrestricted motion and the order parameter (S) becomes zero in this case. Whereas DNA bound ALL faces an exceedingly aligned distribution within both GQ-DNA environments under K+ and Na+ ionic conditions and therefore here the order parameters become 0.256 and 0.433 respectively. We could thus anticipate the angular range of the constrained internal motion of the quadruplex bound ALL from the calculated values of semi-cone angle θ (Scheme 2).

However we have obtained two different set of parameters of ALL internal

motions, like order parameter, diffusion coefficients etc. for the two distinct G-quadruplex DNAs. In Na+, our htel22 sequence forms an antiparallel stranded basket type G-Quadruplex which consists of one narrow, one wide and two medium grooves. Here the loops are edgewisediagonal-edgewise5. Most probably here ALL resides within one of the grooves (wide, narrow and medium) and possibly fenced by sugar backbone which resulted electrostatic interaction further restricting its rotational motion within the groove. Otherwise ALL could also be accommodated either in diagonal loop or edgewise loop along with end-staking on external quartets which originates a restrictive force on the alkaloid. While in K+ solution htel22 sequence gets stabilized to mixed-hybrid quadruplex structure containing one double-chain reversal and two edgewise loops along with one wide groove, two medium sized grooves, one narrow groove.



22

Here the grooves are generally guarded by the double chain reversal loops which make either the edge groove or double-chain-reversal loop and/or lateral loops the favorable binding sites for ALL. Nevertheless, groove size is another determinant factor for the ligands to fit inside it56. Therefore, in this case, ALL might be collaborating with the loops of the hybrid GQ-DNA either via staking or creating H-bond on one/two loop bases accompanied by the poor fractional end staking at the closing part of the G-quartet. However, the corresponding affinity of this binding mode is much lower in comparison to the end staking or sandwich staking or groove binding. The flexibility of the loops and the limited - staking interface are the reasons behind this low affinity57. For the mixed-hybrid (3+1) quadruplex structure, as discussed above, the alkaloid was possibly attached in the loop and/or grooves along with partial end staking which is revealed by the order parameter (S=0.256), diffusion constant (D=2.62 x108), and angular range (θ =59.220) (Table4)58. In contrast, ALL was possibly accommodated within antiparallel basket type GQ-DNA in one of its grooves (wide, narrow and medium) and possibly fenced by the sugar backbone leading to the hugely restricted diffusion of ALL inside G-quartet groove. This in turn get reflected by the higher order parameter (S =0.433), the lower diffusion constant (D=0.85 x108) and narrower angular range (θ=29.950) compared to the other GQ-DNA topology in K+ ions (Table 4)58. Hence, our investigation shows that the binding affinity becomes higher when the binding mode is more restrictive. We could therefore consider the order parameter (S) as a substitute in evaluating the selectivity of the ligand for GQ-DNA. Altogether, our time-resolved fluorescence anisotropy results have been interpreted successfully using wobbling-in-cone model.


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23

Molecular Docking. Having information about the probable binding site inside the DNA is truly crucial in predicting the efficiency of a biologically active drug molecule in the medicinal application. The molecular docking calculations allow us to estimate the binding location of the ligand in the DNA environment. Figure 8 shows the most probable docking pose depicting the binding of ALL with mixed hybrid GQ-DNA in K+ condition and antiparallel GQ-DNA under Na+ ionic condition. The binding stance indicates that the ligand ALL undergoes loop binding through TTA bases of the mixed-hybrid GQ-DNA via hydrogen bonding and van-der-Wal interaction. The specific region is highlighted in the Fig.8a. In the case of antiparallel quadruplex DNA, ALL is attached through medium groove binding and fenced by the sugar backbone. The stance is shown in Figure 8b.Here this is a conceivable binding mode because ALL is unlikely to have end staking due to its nonplanar structure. Thus docking results are consistent with the above experimental findings. IV. CONCLUSION Here, by using various spectroscopic techniques, the interactions of natural alkaloid ALL with the two different GQ-DNA topologies mixed-hybrid under K+ ions and basket-type antiparallel under Na+ condition have been extensively studied. Our steady state and time-resolved study revealed predominant involvements of static type quenching mechanism in both the interactions due to ground state complex formation with the negligible dynamic contribution. Binding constants and number of binding sites hints that ALL has stronger affinity to the anti-parallel Gquadruplexes compared to the mixed-hybrid GQ-DNA. Our FID assay results also exhibits higher selectivity for basket-type anti-parallel GQ-DNA compared to the mixed-hybrid quadruplex structure. Moreover, ALL shows modest selectivity for both the quadruplexes over duplex DNA. Possibly the tertiary nitrogen and carbonyl group of ALL is facilitating hydrogen



24

bonding, van-der-wal interaction, π-π staking with the bases of both GQ-DNAs and also favoring the electrostatic interaction with the phosphate backbone of DNAs. FID results also suggests that during the binding of ALL with both GQ-DNAs, ALL undergoes an indirect fight to bind in grooves and/or loops; clearly distinct from usual -staking on GQ-DNAs’ exterior quartets. We have found modest stabilization of both GQ-DNA topologies upon binding with ALL. The differences in the dynamics of ALL with in both GQ-DNA environments have been demonstrated thoroughly via wobbling-in-cone model in the time-resolved anisotropy analysis. This analysis provides us with a fair account of the dynamics of the constrained inner rotation of the alkaloid inside the two GQ-DNA environments. The alkaloid was binding with the antiparallel G-quartet possibly by partial end-staking and groove binding with high affinity. On the other hand, ALL binds to the mixed hybrid structure moderately in the loops of DNA along with fractional end-staking. Our docking results further confirmed the above findings. Hence, our present investigation has shown that the small planar natural alkaloid ALL binds in two completely different modes with the two different GQ-DNA structures antiparallel and mixed hybrid with different affinity. Thus, ALL could able to discriminate two different GQ-DNA structures. This could be further exploited in the development of the therapeutic agents in targeting particular G-quadruplex DNA conformation. Additionally, present investigation could be extended in the development of the other derivatives of ALL with improved quality of selective targeting of GQ-DNA in cancer cells and with greater stabilization properties which are expected to contribute in the emergence of novel anti-cancer therapies with diminished toxicity. Supporting Information. Figure of the optimized geometry of the molecule ALL (Fig.S1), Fluorescence Lifetime Decay Curve of ALL in the presence of increasing concentrations of (a) GQ-DNA under K+ condition, and (b) GQ-DNA under Na+ condition (Fig.S2a&b), Table of the


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25

fluorescence lifetimes of ALL in the free form and ALL- GQ-DNA complexes (Table S1), Table of Comparative FID results for ALL (Table S2), and Fitted parameters of anisotropy decays of ALL and ALL-GQ-DNA complexes in the presence of K+ and Na+ ions (TableS3). ACKNOWLEDGEMENTS We are heartily thankful to Prof. Nikhil Guchhait of Chemistry Department, University of Calcutta, Kolkata, for providing the opportunity to handle the time-correlated single-photon counting machine. PM is grateful to Science and Engineering Research Board (SERB), New Delhi, India for granting the financial assistance in the form of grants and fellowships (Project File No. SB/YS/LS-199/2013).



26

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& Med.Chem., 2016,24, 33-41. 54. Monchaud, D.; Allain, C.; Bertand, H.; Smargiasso, N.; Rosu, F.; Gabelica, V.; De Cian, A.; Mergny, J.-L.; Teulade-Fichou, M.-P. Ligands Playing Musical Chairs with GQuadruplex DNA: A Rapid and Simple Displacement Assay for Identifying Selective GQuadruplex Binders, Biochimi, 2008, 90, 1207-1223. 55. Paul, B.K.; Guchhait, N. Modulation of Prototropic Activity and Rotational Relaxation Dynamics of a Cationic Biological Photosensitizer within the Motionally Constrained Bio Environment of a Protein, J.Phys.Chem. B, 2011, 115, 10322-10334. 56. Wang, Y.; Patel, D. J. Guanine Residues in d(T2AG3) and d(T2G4) form Parallel-Stranded Potassium Cation Stabilized G-Quadruplexes with Anti Glycosidic Torsion Angles in Solution, Biochem., 1992,31,8112-8119. 57. Luo, D.; Mu, Y. All-Atomic Simulations on Human Telomeric G-Quadruplex DNA Binding with Thioflavin T, J.Phys.Chem.B, 2015, 119, 4955–4967. 58. Jia, G.; Feng, Z.; Wei, C.; Zhou, J.; Wang, X.; Li, C. Dynamic Insight into the Interaction between Porphyrin and G-quadruplex DNAs: Time-Resolved Fluorescence Anisotropy Study, J. Phys. Chem. B, 2009, 113,16237-16245.



36

Figure Captions: Figure 1. (a) Different Structures of GQ-DNA (b) Allocryptopine (ALL). Figure 2. Absorbance spectra of Allocryptopine (ALL) (Conc. ~ 100µM) in the presence of increasing GQ-DNA concentrations in K+ condition from 0-8µM (inset: GQ-DNA concentrations in Na+ condition from 0-8 µM. Figure 3.

Emission Spectra of Allocryptopine (50µM) in the presence of increasing

concentrations of (a) GQ-DNA under K+ condition from 0,1,2,3,4,5,6,7,8 µM (inset: SV plot of ALL-GQ-DNA interaction nunder K+ condition) and (b) GQ-DNA under Na+ condition from 0,1,2,3,4,5,6,7,8 µM (inset: SV plot of ALL-GQ-DNA interaction under Na+ condition), (c) Plot of log[(F0 –F)/F] vs log[GQ-DNA] for the interaction of ALL and GQ-DNA in the presence of Na+ ions.(Inset: Plot of log[(F0 –F)/F] vs log[ct-DNA] for the interaction of ALL and GQ-DNA in the presence of K+ ions.). Figure 4. Circular Dichroism (CD) spectra for (a) (3+1) hybrid GQ-DNA (10µM), (b) antiparallel GQ-DNA (10 µM) at 2, 5, 7, 9, 11, 13, 15µM concentration of ALL. Figure 5. CD melting profiles of G-quadruplex (10µM) at 295nm: (a) in the presence of K+(1) no ligand, (2) 10µM ALL, (inset: in the presence of Na+(1) no ligand, (2) 10µM ALL). Figure 6. Fluorescence displacement of TO bound (a) GQ-DNA in the presence of Na+ by 1, 2, 3, 5, 6, 7, 8, 9, 10 µM of ALL, (Inset: TO bound GQ-DNA in the presence of K+ by 1, 2, 3, 5, 6, 7, 8, 9, 10 µM of ALL ), (b) duplex-DNA by 1, 2, 3, 5, 6, 7,9,11,14,16 µM of ALL. Scheme 1.Probable Binding position of Allcryptopine with the antiparallel and mixed hybrid GQ-DNA.


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37

Figure 7. (a) Variation of steady state fluorescence anisotropy of ALL (50µM) as a function of increasing concentration up to 7.5 µM of GQ-DNA in the presence of Na+ (Inset: Variation of steady state fluorescence anisotropy of ALL (50µM) as a function of increasing concentration up to 7.5 µM of GQ-DNA in the presence of K+) (b) Time-Resolved anisotropy decay of ALL (50µM) in the (▪) absence of DNA, (●)presence of GQ-DNA (7.5 µM) under K+,(Inset: TimeResolved anisotropy decay of ALL (50µM) in the (▪) absence of DNA, (●)presence of GQ-DNA (7.5 µM) under Na+) (ex = 285nm, mon = 323 nm). SCHEME 2: Schematic Representation of Time-Resolved Fluorescence Anisotropy of GQuadruplex DNA and ALL Complexes Using Wobbling-in-the-Cone Model. Figure 8. Docked structure of (a) ALL-antiparallel GQ-DNA complex (representative snapshot of groove binding), and (b) ALL-mixed hybrid GQ-DNA complex (representative snapshot of loop binding). ALL is colouredgreen-red-blue-grey in the ball and stick mode. Table 1.Stern-Volmer Quenching constants for ALL-GQ-DNA interactions in the presence of K+ and Na+. Table 2.Binding parameters for ALL-GQ-DNA interactions in the presence of K+ and Na+. Table3.Fitted parameters of anisotropy decays of ALLand ALL-GQ-DNA complexes in the presence of K+ and Na+ ions. Table 4. Calculated parameters of the Wobbling-in-cone model.



38

Fig. 1(a)

Fig.1b


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Page 39 of 49

39

1.5 Absorbance

Absorbance

3

2

1.0 Na+

0.5 0.0

260

280 300 Wavelength/nm

320

1 K+

0

260

280 300 Wavelength/nm

320

Fig.2

200000 1.8 1.6

160000

F0/F

Relative Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.4 1.2

120000

1.0 0.000000

0.000004

0.000008

[Q]

80000

K+ 40000 0 300

325

350

375

Wavelength/nm Fig. 3(a)


400

425


40

3.5

210000

3.0 2.5

F0/F

Relative Intensity

180000 150000

2.0 1.5

120000

1.0 0.000000

90000

0.000004

0.000008

[Q]

Na+

60000 30000 0 300

325

350

375

400

425

Wavelength/nm Fig.3(b)

0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2

Na+

0.00 -0.25 log[F0-F/F]

log[(F0-F)/F]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 49

-0.50

K+

-0.75 -1.00 -6.0 -5.8 -5.6 -5.4 -5.2 -5.0 log[Q]

-6.0 -5.8 -5.6 -5.4 -5.2 -5.0 log[Q] Fig.3c


Page 41 of 49

41

4

CD[mdeg]

3 +

2

K

1 0 -1 -2 225

250

275

300

325

350

275 300 325 Wavelength/nm

350

Wavelength/nm Fig 4a

CD[mDeg]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3 2 1 0 -1 -2 -3 -4 -5

Na+

250

Fig.4b



1.0 0.8

K+ 2

0.6 0.4 0.2

1 Normalized CD(mdeg)

Normalized CD (mdeg)

42

1.0 0.8

Na+

0.6

1 2

0.4 0.2 0.0

0.0

20 30 40 50 60 70 80 90 Temperature (0 C)

20 30 40 50 60 70 80 90 Temperature (0C) Fig.5

100

120 Relative Intensity

Relative Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60

100 80 60 40

K+

20 0

525

40 20 0

550

575

600

625

650

Wavelength/nm

Na+

525

550 575 600 625 Wavelength/nm Fig.6a


650

Page 43 of 49

43

250

Relative Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ds-DNA

200 150 100 50 0

525

550 575 600 625 Wavelength/nm

Fig.6b

Scheme 1


650


44

0.045

Na+

0.035

0.028 0.026

0.030

0.024

Anisotropy(r)

Anisotropy (r)

0.040

0.025

K+

0.022 0.020 0.018 0.016 0.014 0.012 -1

0.020 -1 0

0

1

2

3

4

5

6

7

8

[GQ-DNA]/M

1

2 3 4 5 6 [ GQ-DNA] M

7

8

Fig.7a

ALL ALL+ anti-parallel GQ-DNA

0.6

0.6 r(t)

0.4

0.4

0.2 0.0

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 49

28

0.2

30

32

34

36

38

Time/ns

0.0 ALL ALL+ mixed hybrid GQ-DNA

-0.2 28

32

36 Time/ns

40

Fig.7b


44

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45

Scheme 2

Fig. 8a



Page 46 of 49

46

Fig.8b Table 1 KSV(L.mol-1)

kq(L.mol-1. s-1)

R2

K+

1.003 x 105

4.23 x 1013

0.99188

Na+

3.44 x 105

1.45 x 1014

0.97742

System ALL + GQ-DNA

Table 2 Kb(L.mol-1)

n

R2

in K+

7.74 x 104

0.97912

0.9867

in Na+

2.44 x 108

1.55

0.9949

System ALL + GQ-DNA


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47

Table3. Sample ALL (50µM) In Buffer K+ + 8 µM GQ-DNA Na+ + 8 µM GQ-DNA

PreExponential Factor()

2

1 0.93

Rotational relaxation time(slow) (ns) 4.63

0.07

1.0723 0.9973

0.81

1.85

0.19

0.9196

Rotational relaxation time(fast) (ps) 608 603

Preexponential Factor(β)

623

Table 4 Sample

r/ns

ALL ALL+ 8 µM GQ DNA in K+ ALL+ 8 µM GQ-DNA in Na+

Dr/s-1

Anisotr opy

0.608 4.635

Hydrodynamic Radius (Å) 9.121 17.94

2.74x108 3.59 x107

0.263 0.273

1.85

13.22

9.01 x107

0.338


/ns

D/s-1

θ (deg)

S

0.693

8

2.62 x10

59.22

0 0.256

0.94

0.85 x108

29.95

0.433


48

TOC GRAPHIC


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Sensing of Different Human Telomeric G-Quadruplex DNA Topologies

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