Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling

Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling

Journal Pre-proof Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectro...

7MB Sizes 0 Downloads 4 Views

Journal Pre-proof Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling

Puja Paul, Soumya Sundar Mati, Gopinatha Suresh Kumar PII:

S1011-1344(19)30794-8

DOI:

https://doi.org/10.1016/j.jphotobiol.2020.111804

Reference:

JPB 111804

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date:

20 June 2019

Revised date:

18 January 2020

Accepted date:

21 January 2020

Please cite this article as: P. Paul, S.S. Mati and G.S. Kumar, Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling, Journal of Photochemistry & Photobiology, B: Biology(2020), https://doi.org/10.1016/j.jphotobiol.2020.111804

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof

Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling

Puja Paul,†§ Soumya Sundar Mati¶ and Gopinatha Suresh Kumar§* †Department of Chemistry, Dinabandhu Mahavidyalaya, Bongaon, West Bengal 743235,

of

India

CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India

na

Address for Correspondence:

lP

re

-p

§

ro

¶ Government GD College, Keshiary, Paschim Medinipur, West Bengal 721135, India

Dr. G. S. Kumar

ur

Scientist

CSIR-Indian Institute of Chemical Biology

Jo

Kolkata 700 032, INDIA Tel: +91 33 2499 5723

e-mail: [email protected] /[email protected] *corresponding author

1

Journal Pre-proof Abstract The ubiquitous influence of double stranded RNAs in biological events makes it imperative to gather data based on specific binding procedure of small molecules to various RNA conformations. Particular interest may be attributed to situation wherein small molecules target RNAs altering their structures and causing functional modifications. The main focus of the study is to delve into the interactive pattern of two small molecule phenothiazinium dyes methylene new methylene blue with three duplex RNA polynucleotides-poly(A).poly(U),

of

blue and

ro

poly(C).poly(G) and poly(I).poly(C) by spectroscopic as well as molecular modeling techniques.

-p

Analysis of data as per Scatchard and Benesi-Hildebrand methodologies revealed highest affinity

re

of these dyes to poly(A).poly(U) and least to poly(I).poly(C). In addition to fluorescence quenching, viscometric studies also substantiated that the dyes follow different modes of binding

lP

to different RNA polynucleotides. Distortion in the RNA structures with induced optical activity

na

in the otherwise optically inactive dye molecules were evidenced from circular dichroism results. Dye-induced RNA structural modification occurred from extended conformation to compact

ur

particles visualized by atomic force microscopy. Molecular docking results revealed different

Jo

binding patterns of the dye molecules within the RNA duplexes. The novelty of the present work lies towards a new contribution of the phenothiazinium dyes in dysfunctioning double stranded RNAs, advancing our knowledge to their potential use as RNA targeted small molecules. Keywords: Phenothiazinium dye, Double-stranded RNA, Spectroscopy, Intercalation, Modeling

2

Journal Pre-proof

1. Introduction Emerging interest biophysical research has aroused owing to the potential of RNA to function as convenient binding platforms, in the realm of RNAi and in the elucidation of gene expression pathways [1]. Deleterious effect on DNA has been a commonly examined zone which promotes carcinogenesis leading to genetic disorders and cancer development. On the contrary, due to scanty understanding of RNA functional behavior, potential triggers of RNA damage posing

of

lethal insults to cell remain at a rudimentary level even today. RNA damage actively contributes

ro

to the onset of many known diseases like Parkinson, Alzheimer, dementia with Lewy bodies, etc

-p

[2]. Being the genetic material of a number of viruses, RNA is acknowledged as the key activator

re

against viral infections and RNA viruses are known to exacerbate asthma, COPD and other

lP

respiratory diseases. The potential biotechnological applications of RNA-triggered sequencespecific degradation of a homologous mRNA are spurring new avenues by providing advances in

na

studies on RNA damage.

The inability of certain functional RNAs to be translated into a protein give rise to duplex type

ur

double stranded RNAs (dsRNA), the phenomenon described as RNA interference [3]. Several

Jo

dsRNA like 16S rRNA gene, U1, U2, U4 snRNAs, M1 RNA (from E. coli RNase P), mitochondrial introns, tRNA function as molecular marker in microbial ecology, have roles in pre-mRNA splicing and in-cell solubility of C5 protein, gene specific silencing, form selfsplicing RNA molecules, etc [4-8]. RNAs assume a plethora of complex secondary and tertiary structures, in trans and cis conformation, differing in base-pairing modes, spanning from Watson-Crick to different canonical forms [9]. Due to low availability of various sequences of dsRNA, small molecules binding study with dsRNA based on spectroscopic parameters are limited; the common being polyadenylic-polyuridylic acid

3

[poly(A).poly(U)], polycytidylic-

Journal Pre-proof polyguanylic acid [poly(C)·poly(G)] and polyinosinic-polycytidylic acid [poly(I).poly(C)] [3,1219]. The metabolically stable double-stranded complex of synthetic RNA, poly(A).poly(U) act as an IFN-inducer, has been exploited for treating breast cancers that express toll-like receptor 3 (TLR3), etc [20,21]. Poly(I).poly(C) can interact with melanoma differentiation-associate gene 5 (MDA-5),

induce

activation

of

NF-kβ

and

can

inhibit

estradiol synthesis

[22,23].

of

Poly(C)·poly(G) demonstrate similar type of interferon-inducing activity when administered to

ro

white mice as poly(I).poly(C) [24].

-p

The two dyes, belonging to phenothiazinium group, methylene blue (MB) and new methylene

re

blue (NMB), are structurally close in their basic phenothiazinium skeleton; NMB bears two methyl substituents at 2, 8 positions (Fig. 1). MB significantly inhibits antidepressant target oxidase (MAO) and

its association to the chronic neurodegenerative disease

lP

monoamine

na

Alzheimer's disease has aroused profound interest [25]. MB, due to its vasoconstrictive effects, prohibits smooth muscle relaxation and

vasodilation by hindering the cyclic guanosine

ur

monophosphate pathway controlled by nitric oxide [26]. High levels of potentially fatal serotonin

Jo

accumulate in the brain of psychiatric patients, if administered with MB, due to its monoamine oxidase inhibitor (MAOI) property [27]. NMB is a hazardous water pollutant, being extensively used in textile and coating industry, when flushed out can cause serious consequences affecting fish and other aquatic life [28] Dilute solutions of NMB produce little gastrointestinal distress and may cause weakness, muscular twitching. Biophysical approach coupled with molecular modeling studies were employed here for understanding the pattern of interaction of these dyes with model ds RNA structures. The novelty

4

Journal Pre-proof of the present in vitro studies results on the structural and conformational aspects which may be correlated to RNAs cellular dysfunction. This, in turn, would advance our knowledge towards possible disorders imposed on RNA structure on account of exposure to these dyes; benefitting the arena of designing RNA targeted drugs for therapy.

Experimental Section 2.1.Materials

of

Poly(A).poly(U), poly(I).poly(C), and poly(C).poly(G) were procured from Sigma-Aldrich

ro

Corporation with characteristics detailed elsewhere [29]. Concentration of these duplexes (in

-p

base pairs) were spectrophotometrically measured using known molar absorption coefficient (ε)

re

values [10,13,29] Uniform size of about (280 ±50) base pairs of the RNAs was ensured by sonication in a Labsonic unit [13].

lP

Buffer used for the experiments was 20 mM Na+ cacodylate, pH 7.2, prepared in deionized and

na

doubled distilled water. 2.2. Preparation of dye solutions

ur

MB (CAS No. 122965-43-9, CI: 52015, purity  85%), NMB (CAS No. 1934-16-3, CI: 52030,

Jo

purity  70%) (dyes hereafter) were also products of Sigma-Aldrich Corporation. Spectrally purified dyes (> 98%) were obtained on alumina using carbon tetrachloride by column chromatography after subsequent drying. The concentration was estimated using ε as : MB 76,000 M-1. cm-1 at 664 nm NMB - 50,400 M-1. cm-1 at 630 nm. Fresh dye solutions prepared in the experimental buffer were kept protected in the dark until used. The dye concentration in each experiment was kept at the lowest possible to avoid aggregation and adsorption on the cuvette walls. Beer’s law was obeyed in the concentration range used here. 2.3. Absorption and fluorescence spectral studies and elucidation of binding parameters 5

Journal Pre-proof A Jasco V 660 spectrometer (Jasco, Japan) was utilized for absorbance studies at (20±0.5)° Celsius. This unit was equipped with a thermoelectrically controlled cell holder and temperature controller. Matched quartz cuvettes (10 mm path, Hellma-Germany) were used [13]. Steady state fluorescence was performed using a Shimadzu RF-5301PC unit (Shimadzu, Japan) in 1 cm path length cuvettes [13]. MB and NMB were exited at 610 and 635 nm, respectively, and the emission intensity was monitored in the range 620-750 nm maintaining an excitation and

of

emission band pass of 5 nm. In practice, a defined concentration of the dye was mixed with

ro

varying concentrations of RNA to generate various P/D (RNA nucleotide phosphate/dye molar)

-p

ratios. The titrations were performed until we observed saturation. Usually 5 min. equilibration

re

time was given after each addition of titrant solution into the sample. The control cuvette contained the same amount of polynucleotide as the sample cuvette.

lP

Data obtained were profiled into Scatchard plots (r/C f versus r). This robust procedure provides

na

an unambiguous method of estimating the affinity ligand-RNA interactions under equilibrium condition. The plots yielding (+) slope at low r values were analyzed using McGhee-von Hippel

ur

protocol for cooperative binding [13,30]

(1)

Jo

r/Cf = K(1-nr)×[{(2+1)(1-nr)+(r-R)}/2(-1)(1-nr)](n-1)[{1-(n+1)r+R}/2(1-nr)]2 where, R = {[1-(n+1)r]2 + 4r(1-nr)}1/2

Here, K is the intrinsic binding constant to an isolated binding site, ‘n’ is the number of base pairs excluded by the binding of a single dye molecule and ω is the cooperativity factor. The binding ratio r is the number of moles of ligand bound per mole of nucleotide. It is defined as r = Cb / [RNA]total where Cb is the concentration of the bound dye and Cf is the concentration of the free dye. Origin regular 7.0 version software (Microcal Inc., acquired by Malvern Instruments) determined the best-fit parameters of K and ‘n’ to the above relation. 6

Journal Pre-proof Scatchard plots of NMB-poly(C).poly(G) and NMB-poly(I).poly(C) displayed negative slope at low r values indicating non-cooperative binding. Therefore, these were analyzed using the following McGhee-von Hippel equation for non-cooperative binding [30], r/Cf = K (1-nr)[(1-nr)/{1-(n-1)r}](n-1)

(2)

Here, K is the intrinsic binding constant to an isolated binding site, and ‘n’ is the number of base

of

pairs excluded by the binding of a single ligand molecule.

ro

Benesi–Hildebrand (BH) plots were also constructed utilizing the titration data by using the

-p

equation, 1/∆A = 1/∆Amax + {1/KBH(∆Amax )}×1/[M]

(3)

re

Here, [M] is the concentration of the RNA and ∆A is the difference in absorbance of

lP

uncomplexed and complexed dye at max . By plotting 1/ absorbance intensity with respect to 1/

na

[RNA], the Benesi-Hildebrand association constant for the complex formation (KBH) was derived from the ratio of the slope/intercept [31]. Several scenarios of such binding outcomes have been

ur

observed previously also [3,10,11,13,14,17,18].

Jo

2.4. Determination of binding stoichiometry Averaged from at least three experiments, the binding stoichiometry was determined by continuous variation method (Job) [32] from fluorescence spectral data as reported in the literature [33]. The stoichiometry was obtained in terms of RNA-dye [(1-χdye)/ χdye] where χdye denote the mole fraction of the dyes. 2.5. Fluorescence quenching studies Quenching protocol utilized the anionic quencher [Fe(CN)6 ]4-. Different ratios of potassium chloride and potassium ferrocyanide solutions were mixed to obtain changing concentration of

7

Journal Pre-proof the ferrocyanide as outlined earlier [13]. The results were plotted as Stern-Volmer plots of (Fo /F) versus ferrocyanide ion concentration [13]. 2.6. Solution viscosity measurement In viscosity experiments the time needed to flow through the semi-micro 75 size capillary viscometer (Cannon, USA) that was kept vertically in a bath maintained at (20±1)° Celsius was

specific relative viscosity was calculated as per the relation, η´sp /ηsp = {(tcomplex – to )/to }/{(tcontrol – to )/to }

of

measured. Detailed experimental procedures are reported in the literature [34]. The relative

ro

(4)

-p

where, η´sp and ηsp are specific viscosities of the dye-RNA complexes and RNA, respectively;

respectively.

lP

2.7. Circular dichroism measurements

re

tcomplex , tcontrol, and to are flow times (average) for the dye-RNA complexes, RNA and buffer,

na

CD spectral measurements employed a Jasco J815 unit interfaced with a Jasco temperature controller as described [11,35]. Equipment settings were: scan rate of 100 nm/min., band-width

ur

of 1.0 nm, response time of 1 sec. and sensitivity of 100 milli degree, no of scans = five. The

Jo

spectra were plotted as in terms of molar ellipticity [θ] in terms of either per RNA base pairs (210-400 nm) and per bound dye (300-700 nm). 2.8. Atomic force microscopy (AFM) Preparation of RNA samples and complexes with dyes was done in filtered water for AFM imaging. From a final concentration 10 ng/μL, 10 μL of each sample was aliquoted for adsorption onto a freshly cleaved muscovite Ruby mica sheet (ASTM V1 grade Ruby Mica from MICAFAB, Chennai). This was followed by drying for 20 min in vacuum under nitrogen gas. A final concentration of 10 ng/μL for RNAs and 1 ng/μL for the dyes was maintained for the RNA-

8

Journal Pre-proof dye complexes. Incubation for the mixture was done for 5-10 min followed by similar method of adsorbing onto the mica sheet. Processing and manipulation of images were done through Picoview version 1.1 software and Pico Image Advanced software (Agilent Technologies), respectively following protocol mentioned in details earlier [29]. 2.9. Molecular modeling studies Using density Functional Theory with B3LYP functional, computational calculations for initial

of

geometry optimization of MB and NMB molecules in their ground states with standard basis set,

ro

6-311G(d,p) was done [36-38]. Structures of AU, CG and IC polynucleotides used for docking

-p

study were constructed from three different crystal structures of Protein Data Bank (PDB)

re

identifier 1PLY, 2ANA and 2Q66 [39] and using Abalon software. Other related parameters were kept in same as in previous discussions [40,41]. As known, an intercalator molecule slides

lP

between two consecutive base pairs in such a way that affects the double strand by gap opening

na

between these base pairs. Certainly, our docking simulations with MB and NMB molecules are also targeted in preformed sites. However, when the targeted sequences directly from crystal

ur

structures having lack of prior gap, such sites are unavailable. Consequently, it is needless to say,

Jo

in every cases AutoDock was not able to dock the ligand molecules as intercalators. Even in torsional flexibility as applied in AutoDock, is not sufficient to persuade gap opening between desire base pairs [42]. After failure to reveal gap opening between the desired base pairs in time of docking simulations, we changed our aim into an another alternative by forming an binding site for appropriate intercalative binding prior to docking simulation for such constructed double strand structures. As our endeavor to monitor the gap opening between the relevant base pairs during the docking simulations failed, an alternative method was thought of by forming an intercalation binding site prior to docking. Previously reported energy minimization procedure of

9

Journal Pre-proof gap opening through classical and threading intercalator molecules are placed between two base pairs of AU and CG polynucleotides respectively to get classical and partial intercalator complex [43]. As a result of it, complex was minimized leading to a structure with an intercalation gap which was subjected to docking simulations. After forming the classical and threading intercalation gap in AU and CG polynucleotides respectively and IC as obtained as constructed, in further AutoDock procedure polar hydrogen atoms and Gasteiger charges were included to

of

arrange the three RNA molecules for docking analysis. Ligand docking was performed using

ro

Lamarckian genetic algorithm (LGA) employed in AutoDock 4.2. Grid size was fixed to 120,

-p

120 and 120 along the three axes to identify the ligand binding site into the RNAs.

re

3. Results and discussion

lP

3.1. Electronic absorption spectral monitoring

UV-visible absorption technique can very efficiently determine strength of interaction when a

na

small molecule and bio macromolecule are titrated by correlating the extent of changes in absorbance value and or observing the shift in the position of peaks [44]. max of MB at 664 nm

ur

(with a hump around 620 nm) and NMB at 630 nm (with a shoulder at 591 nm) were selected to

Jo

record any change in the absorption profile on incremental addition of RNA concentrations. The π-π absorption band showed decreasing nature (hypochromic effect) accompanied by 1-2 nm of red-shift (bathochromic effect), indicating the formation of a dye-RNA complex. The dimethyl groups in MB and diethyl groups in NMB hinder the formation of H-bonds between the dyes and free water molecules which is manifested as a slight red shift in the spectral study. The planar dye chromophores may be stacked between the RNA base pairs stabilizing the RNA helix, resulting in bathochromism.

Conjugation due to

the electron rich substituents on the

phenothiazinium ring results in decrease of excitation energy necessary for π-π transition owing 10

Journal Pre-proof to the decrease in absorbance. The partially filled coupling π orbital is responsible for the change occurring in electronic state, thus, decreasing the transition probabilities, which is accompanied by hypochromism [45]. Isosbestic points were observed in the spectral profile in the case of interaction of MB-AU, MBCG, MB-IC and NMB-AU polynucleotide systems at 605 and 686 nm, 686 nm, 683 nm, 570 and 663 nm, respectively,

indicating spectroscopically distinct chromophores, namely, free and

of

bound species at any wavelength showing equilibrating circumstances in the dye-RNA

ro

interaction. Complexation of NMB with both of CG and IC polynucleotides did not present any

-p

isosbestic points which may suggest the lack of a classical intercalation phenomenon. Figure 2

re

a,b are the representative spectral data for MB-AU and NMB-AU interaction. As the binding satisfied equilibrium condition, a constant concentration of each of the RNA polynucleotides was

lP

titrated by adding increasing concentration of dyes for estimating the free and bound dye as

na

documented previously [10]. The binding pattern was quantified by establishing a Scatchard plot of r/Cf versus r based on the spectral changes. The inset of Fig. 2a,b represents the corresponding

ur

Scatchard plots depicting the complexation of the dyes to AU polynucleotide. The bell shaped

Jo

nature of the binding isotherms possessing positive slopes at low levels of binding (low r values) clearly endorses the fact that the dyes prefer cooperative binding mode. This, in turn, is suggestive to fit these plots in accordance with McGhee and von Hippel methodology for a cooperative binding system to derive the intrinsic binding constant (K) and the number of base pairs (n) excluded by the binding of a single dye molecule [30]. The presentation in Table 1 illustrating the binding affinity values, clearly proposed that both the dyes have highest affinity to AU polynucleotides and lowest affinity to the IC polynucleotide. The product of K and the cooperative factor (ω) is the intrinsic binding constant (Ki), which yielded maximum value for

11

Journal Pre-proof MB-AU complexation followed by NMB- AU, MB-CG (Fig. S1a) and MB-IC (Fig. S1b) polynucleotides. An induced allosteric change or effect brought about by a change in the conformational feature of the polynucleotide as a consequence of binding to small molecule provide plausible explanation for positive cooperativity [46]. Charge distribution, which in this case is located on the region of the dye which intercalates between the RNA base pairs and helix flexibility are the important dictating parameters in the cooperative binding mechanism [47]. It is

of

likely that initial binding of the dyes is cooperative which convert the discrete heterogeneous

ro

structures of double stranded RNAs to a structure similar to the conventional A-form where

-p

subsequently the binding becomes non-cooperative [13]. Nevertheless, Scatchard cum McGhee

re

and von Hippel methodology could not evaluate the binding affinity value between NMB-CG (Fig. S1 c) and NMB-IC (Fig. S1 d) due to lack of isosbestic points. As a result of this, binding

lP

constant (KBH) for the complexation process were evaluated by Benesi-Hildebrand (BH)

na

methodology [36,37] [Table 2]. The order of K and KBH are outlined in Table 1 and Table 2. 3.2. Spectrofluorimetric titration studies

ur

The fluorescence intensity of MB and NMB around 680 and 650 nm, respectively, showed a

Jo

regular decrease in its value while no apparent shifting in the maximum emission wavelength occurred on increasing the concentration of RNA duplexes. The dyes, being endowed with electron rich substituents at 3 and 7 positions are involved in π conjugation with the heterocyclic ring which is conducive to a high fluorescence emission. Interaction with RNA increases the rigidity of the flexible substituents inside the helix and the binding capacity between amino substituents and negatively charged backbone of the helix. As a consequence of this, the dyes collide with solvent molecules at a reduced frequency i.e ligand molecules are not fully screened from water molecules resulting in fluorescence quenching [47]. The fluorescence quenching of

12

Journal Pre-proof the dyes by RNA likely does not happen through energy transfer phenomenon, as emission bands of the dyes lie at a higher wavelength as compared to absorbance band of RNA. A representative fluorescence emission pattern of the dyes in the presence of AU (Fig.3a,b) shows the magnitude of quenching to vary in the order as MB-AU polynucleotide (82.01%) > NMB- AU polynucleotide

(74.23%) >

MB-CG polynucleotide

(63.11%) >NMB-CG polynucleotide

(54.33%) > NMB-IC polynucleotide (39.17%) > MB-IC polynucleotide (33.07%). The

of

interaction of the dyes in presence of CG and IC are shown in Fig. S2. This result tend to

ro

propose ligand-ligand excitation interactions in the presence of RNA or changes in ligand

-p

environment, like hydrophobic interactions with the heterogeneous RNA binding sites versus

re

hydrophilic interactions with the solvent molecules [48]. Using Scatchard and McGhee-von Hippel analysis, the binding affinity values derived from spectrofluorimetric analysis were very

lP

similar to those obtained from spectrophotometric analysis (Table 1 and inset of Fig. 3a,b).

na

Benesi-Hildebrand (BH) methodology [31] was also utilized to analyze the data for the determination of binding constant (KBH) of the interaction [Table 2].

ur

3.3. Evaluation of stoichiometry (Job plot)

Jo

The technique of continuous variation analysis (Job plot) governs the binding stoichiometry of the dyes with RNA, which was conducted in fluorescence maintaining a constant temperature. The binding mode was witnessed to be of single type, for all the interactive modes when the difference in fluorescence intensity (ΔF) at 685 nm and 648 nm, respectively, for MB and NMB versus the mole fraction of the corresponding dyes was plotted (Fig. S3). The intersection point of the two opposite slope lines in the graph points towards the mole fraction of the dyes bound during the complex formation. Calculation based on least square fitted lines at χMB = 0.324, 0.287 and 0.249 correlate with stoichiometry values (number of RNA bases bound per MB) of

13

Journal Pre-proof 2.08, 2.48 and 3.01, respectively, for the complexation with AU, CG and IC polynucleotides, respectively. Stoichiometric number of 2.18, 3.82 and 4.78 corresponds χNMB= 0.314, 0.207 and 0.173, for the complexation with AU, CG and IC polynucleotides, respectively. 3.4. Fluorescence quenching study by ferrocyanide ions Fluorescence quenching study serves as a definitive method to probe the availability of the dye molecules to fluorescence quenchers like ferrocyanide ions [Fe(CN)6 ]4−. This would suffer

of

repulsion by the negatively charged phosphate backbone of RNA polynucleotides if the dyes are

ro

deep seated inside the helix core. Compared to intercalation, the dyes binding to the grooves are

-p

exposed to a greater extent to the solvent, increasing the accessibility of the dyes to the quenchers. On calculation, the values of quenching constants for free MB and its complexes

re

were 45.57, 9.46, 16.92 and 35.98 M-1 and the same for NMB were 39.87, 10.99, 20.14 and

lP

34.47 M-1 with poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C), respectively. The dyes

na

were quenched at a higher percentage in the case of poly(A).poly(U) (79.21% with MB and 72.43% with NMB) when compared with MB and NMB complexes with the other two

ur

polynucleotides which were 62.87% and 49.48% with poly(C).poly(G) and 21.04% and 13.54%

Jo

with poly(I).poly(C), respectively. Figure S4 is the representative Stern-Volmer plots for the quenching of fluorescence of the dye-RNA complexes. The inference of the Stern-Volmer quenching constant values propose the location of both the bound dye molecules to be secluded away from the solvent giving indication towards a strong intercalation mode of binding to AU. The Stern-Volmer values for NMB-IC and only NMB being close to each other prompted us to believe that the quencher has considerable access to NMB, which was not in sandwiched condition between the base pairs of IC. The structure of NMB (Fig. 1) features non-planar substituents along with the planar ring. The methyl groups at 2

14

Journal Pre-proof and 8 positions for NMB prohibits the coplanar phenothiazinium ring to exactly slide in between base pairs of dsRNA causing the substituents to go into the minor grooves. As the two dyes show different percentage of quenching with the polynucleotides, it can be inferred that the dyes approach the polynucleotides in different fashion which largely depends on the latter's conformation and sequence of base-pairs. Furthermore the orientation of base pars, electrostatic charge on them etc. also may influence. Thus, the nature of binding for the dyes become

of

apparent from ferrocyanide ion quenching studies - strong intercalation to poly(A).poly(U),

ro

partial or weak intercalation to poly(C).poly(G) and poly(I).poly(C), as evidenced from

-p

spectrophotometric titration.

re

3.5. Viscosity measurements

A reliable hydrodynamic procedure like viscometric technique can to gain detailed insight on

lP

how the dyes are bound to the RNAs. A classical intercalator can slide itself between the base

na

pairs of double stranded RNAs leading to lengthening and local unwinding of the helix resulting in increase in RNA viscosity. The variation of the relative specific viscosity of the complex

ur

(sp /sp ) with changing mole ratio for the complexation (poly(A).poly(U) + dye) showcased an

Jo

increase in specific viscosity of complex with D/P (dye/ RNA nucleotide phosphate molar ratio) (Fig. not shown). Similar type of increase was also encountered when the dye complexed with poly(C).poly(G) (Fig. not shown). In all cases, MB displayed a larger net increase and a steeper change in comparison to NMB, which required a higher D/P for reaching saturation. Only during the addition of the dyes to poly(I).poly(C) solution slight increase in the flow time was observed, which was not as pronounced as for a classical intercalator and indicate towards groove-binding mode. Although this result indicates intercalation for the other interacting partners, their relative extent is not revealed.

15

Journal Pre-proof 3.6. Affirmation of secondary structural changes in RNA conformation Disparity in the chiral confirmation of RNA due to dye-RNA interactions can be analysed with the aid of circular dichroism (CD) spectroscopy. The region 210-400 nm was chosen to monitor the structural perturbations of RNA structure as non-covalent interactions with the dyes alter the intrinsic CD spectral behavior of RNA. The spectra 1 of Fig. 4 a-f are the CD representation of the three RNA polynucleotides displaying the conventional A-form structure identified by a large

of

positive band in the 260-280 nm region and an adjacent weak negative peak in the 230-235 nm

ro

region. The ellipticity of the long wavelength positive band at 268 nm (assigned to base stacking)

-p

of AU, 272 nm of CG and 278 nm of IC polycucleotided decreased on incrementally adding

re

higher concentrations of the dyes with increasing D/P. Furthermore, negative band attributed to helicity registered no appreciable change. Both the dyes registered visible isoelliptic point at 288

lP

nm with AU and at 270 nm with IC polynucleotide which recommends that the structural

na

changes may be interdependent [18]. The reduction of CD signal at 268 nm may correspond to a minor change in number of base pair per turn in RNA helix [49]. The RNA groove widens owing

ur

to the increase in winding angle and this permits a proper position to the ligand at the

Jo

intercalation site. In comparison to NMB, MB registered a higher change of ellipticity with all three RNAs. Modification in the position and intensity of these spectral bands augment possibility of conversion to a different conformation or encounter distortion in native conformation of RNA duplex in alliance with its interaction with the dyes, which otherwise do not manifest any CD changes in the above range. RNA-induced perturbation of these optically inactive dyes was studied in the range 300-700 nm to further validate the conformational aspects linked to dye-RNA interaction. As the range under investigation lacks any CD contribution from RNAs or dyes, it is anticipated that the

16

Journal Pre-proof chromophore of the achiral dyes may exhibit an induced CD in the chiral domain of RNA. Fig. S5 exemplifies the induced CD spectral changes of MB and NMB when interacted with AU, CG and IC sequences which were obtained by titrating dyes of fixed concentration (30 µM) in presence of incremental addition of the RNAs. A bisignate type induced CD spectra was the outcome of the interaction pattern with the dyes giving rise to two prominent peaks for AU polynucleotide; 610 and 590 nm are for the positive ones while the negative ones were centered

of

at 564 and 549 nm, respectively, for MB and NMB, showing decrease in the ellipticity with

ro

increase in P/D. Similarly, for CG polynucleotide, the positive peaks were evident at 548 and

-p

629 nm and corresponding negative peaks at 508 and 570 nm, respectively, with MB and NMB.

re

The dyes, on interaction with IC polynucleotide apparently decreased the ellipticity of the positive bands (613 for MB and 557 nm for NMB) and a negative band (568 for MB and 595 nm

lP

for NMB) until saturation was reached. The exciton type splitting seen in Fig S5f could be

na

proposed to be due to the emergence of a left-handed helical organization of the dye chromophores which leads to interaction of the transition dipoles of the dye chromophore with

ur

the transition moment of the chirally disposed RNA base pairs [50]. There was observable

Jo

inequality in the strength of binding and location of the dyes inside the double helical organization of the polynucleotides. The base pairing strategy and dipole moments of AU, CG and IC polynucleotides being different, different CD patterns for different interacting partners were obvious. The opposite negative-positive spectral bands may suggest parallel and perpendicular alignment to the base pair axis of RNA. This also provides knowledge regarding the approach of dye molecules to RNAs from both the groove sides and the induced CD signals are expected to appear from the stacked dyes at two sites on the polynucleotide. Nevertheless, isothermal titration calorimetric experiment (not shown here) did not disseminate any affirmation

17

Journal Pre-proof towards two site binding of the dyes to these RNA duplexes. Previously recorded data suggest almost identical bisignate induced CD pattern when safranine O binds with RNA duplexes showcasing a positive peak at around 460 nm, negative peak at around 492 nm and crossover at 477 nm [19]. Based on this, it can be said that MB disrupt interaction between RNA bases causing a higher modification of the RNA structures compared to NMB and hence concurs with other spectroscopic experiments.

of

3.7. Atomic force microscopy: Structural modification caused by the dyes

ro

In situ AFM imaging can predict the nature of dye-dsRNA interaction that can significantly alter

-p

RNA molecular structures [29]. Any unwanted morphological changes induced by the dyes

re

could hold the key for the early detection of disease. Intrigued by the observed changes in CD studies, we attempted to visualise any dye induced high resolution conformational changes of

lP

dsRNA which open the prospect for application of dyes interfering with RNA structure and

na

function. Figure 5a represents the AFM imaging pattern of free poly(A).poly(U) covering a scan area of (400 nm × 400 nm). Figure 5a is the three-dimensional representation of Fig. 5a.

ur

Poly(A).poly(U) images displayed some elongated worm-like structures whose variation in

Jo

length was between (82.00 ± 1.60) nm to (112 ± 1.50) nm. AFM allowed the unambiguous identification of binding of the dyes to AU as their interaction caused deformation in its elongated structure to more circularized one. Figure 5 b,c represent images of AU-MB and AUNMB complex within scan areas of (550 nm × 550 nm) and (300 nm × 300 nm), respectively, while Fig. 5 b,c are the three-dimensional representation. Careful analysis of AFM images of poly(C).poly(G) molecules [Fig 5d] appeared to be condensed bead-like at saturating concentration of the dyes. The average diameters of the CG-MB [Fig 5e] and CG-NMB [Fig 5f] complexes were (58.00 ± 1.85) nm and (46.00 ± 1.45) nm, respectively, and average height was

18

Journal Pre-proof 4.38 nm and 4.24 nm, respectively. Progressive globularization of IC polynucleotide [Fig 5g] as induced by MB and NMB was evident from Fig. 5h and i covering scan areas of (2.22 μm × 2.22 μm) and (1.3 μm × 1.3 μm), respectively . Compactness of the circularized structure reduced and became little bit diffused during complexation of IC polynucleotide with NMB. An expected hydrophobic interaction between the dyes and RNA gave impetus for the conversion of elongated rod like RNAs to coiled ones. This reduction in surface area in contact with water is

of

mainly attributed in minimizing any adverse effect towards the formation of a stable condensed

ro

structure. Structural deformation of biomolecules as a result of the dyes enables us to track the

-p

actual potential of the dyes as causative factors for RNA damage.

re

3.8. Molecular modeling

Molecular modeling study provides complementary insight for monitoring dye-RNA interaction

lP

and identifying ligand binding sites. In search of that modeling study has been performed with

na

two dye molecules MB and NMB into the three double stranded RNA polynucleotides. The results specify the classical intercalation of planar phenothiazinium moiety of MB and NMB to

ur

the intercalation site of double stranded AU polynucleotide.

From molecular modeling

Jo

simulation (Fig. 6), it was seen that MB and NMB are theoretically capable of intercalating within poly(A).poly(U) similar with the experimental findings. It is worth mentioning that the intercalating ability of the whole molecule is increased by decreasing spatial hindrance in the area around the sliding moiety. Clear prediction from Table 3 suggests that the binding energy (30.1 kJmol-1 ) is favorable in MB-poly(A).poly(U) binding than that of binding with NMB (-27.5 kJmol-1 ) showing the highest binding energy value for less branching MB molecule with respect to NMB. In intercalation, the planar phenothiazinium moiety of both MB and NMB molecules are entrapped and slide between two base pairs adenine and uracil of the double stranded AU

19

Journal Pre-proof polynucleotide. This binding scenario is quite different when the dyes bind with CG sequences. From Fig. 6 one can see the partial intercalation of both MB and NMB through the groove of the double strand base pairs. In this partial intercalation some portion of MB and NMB molecules takes place between guanine and cytosine base pairs while the remaining portion extend into the minor groove of CG sequences. The lesser binding energy of the dye molecules in CG sequences compared to AU sequences is also an evidence for partial intercalation. On the other hand the

of

MB and NMB molecules could fit well into the minor groove of IC polynucleotide among the

ro

three I-C base pairs. As in this double stranded sequence the dye molecules resides preferentially

-p

in the minor groove the binding energy is less in this type of binding than that of the previous. It was perceived that binding of the dye molecules was stabilized by the Hydrogen bonds (H-bond)

re

with the polynucleotide bases in addition to van der Waal’s interactions. From closer view of

lP

Fig. 6, it can be seen that the intercalation gap is much more in AU complexation with MB and

na

NMB than that of with CG sequences. In case of classical intercalation, the MB and NMB molecules are inserted in planar orientation through the base pairs of AU whereas in threading

ur

intercalation dye molecules are partially tilted to form partial intercalation with CG. So the above

Jo

discussion throws light on ligand binding efficacy which decreases from MB to NMB. Comparing the binding site and binding location of the two dye molecules inside the polynucleotide bases MB and NMB managed themselves in an intercalative insertion mode into AU whereas in CG and IC polynucleotides their orientations were partial intercalative and groove binding fashion.

4. Conclusions Major structural changes in RNAs appear to play key roles in several chronic diseases. The results obtained here renders deeper acumen into the essence of interaction between the two

20

Journal Pre-proof phenothiazinium dyes, methylene blue and new methylene blue with double stranded RNA. The results, based on spectroscopic and molecular modeling findings have divulged that these dyes bind with three sequence specific RNA duplexes, with remarkably higher binding to the adenineuridine sequences. The inclusion of a bulky substituent on the phenothiazinium moiety of NMB causes reduction in its binding potential in comparison to MB. Based on fluorescence quenching studies and viscosity experiments, the mode of binding to the AU sequences was found to be

of

strong intercalative interaction while weak intercalation scenario was observed in the case of CG

ro

and IC sequences. Results from circular dichroism indicate conformational distortion of RNA

-p

structure with optical activity being induced to the bound dyes but to different extents. AFM

re

imaging illustrated the architectural changes linked to RNA-dye complexes from elongated structures to bead-like forms. Docking study reiterated the intercalative, partially intercalative

lP

and groove binding mode of dye molecules with AU, CG and IC sequences, respectively. The

Acknowledgements

na

results further advance our fundamental understanding towards small molecule-RNA interaction.

ur

Dr. P. Paul was a CSIR SRF at CSIR-IICB at the initial stage of this work. Authors are thankful

Jo

to all the colleagues at both CSIR-IICB & Jadavpur University for help and support during the course of this work. GSK received funding through the CSIR FYP project (BSC0123).

21

Journal Pre-proof References [1] C. Davis-Vogel, A. Ortiz, L. Procyk, J. Robeson, A. Kassa, Y. Wang, E. Huang, C. Walker, A. Sethi, M.E. Nelson, D.G. Sashital, Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm, Sci. Rep. 8 (2018) 7858. [2] A. Xie, J. Gao, L. Xu, D. Meng, Shared mechanisms of neurodegeneration in Alzheimer's disease and Parkinson's disease, Biomed. Res. Int. 2014 (2014) 648740. [3] S.R. Chowdhury, M.M. Islam, G.S. Kumar, Binding of the anticancer alkaloid sanguinarine to double stranded RNAs: Insights into the structural and energetics aspects, Mol. BioSyst. 6 (2010) 1265-1276.

of

[4] L. Vukovic, H.R. Koh, S. Myong, K. Schulten, Substrate recognition and specificity of double-stranded RNA binding proteins, Biochemistry 53 (2014) 3457-3466.

ro

[5] K-N. Son, Z. Liang, H.L. Lipton, Double-stranded RNA is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negativestranded RNA viruses, J. Virol. 89 (2015) 9383-9392.

re

-p

[6] R. Srinivasan, U. Karaoz, M. Volegova, J. MacKichan, M. Kato-Maeda, S. Miller, R. Nadarajan, E.L. Brodie, S.V. Lynch, Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens, PLOS ONE 10 (2015) e0117617.

lP

[7] J. Guiro, S. Murphy, Regulation of expression of human RNA polymerase II-transcribed snRNA genes, Open Biol. 7 (2017) 170073.

na

[8] A. Son, S. Choi, G. Han, B. L. Seong, M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding, RNA Biol. 12 (2015) 1198-1208.

ur

[9] O. Rosok, M. Sioud, Systematic identification of sense-antisense transcripts in mammalian cells, Nat. Biotechno J. 22 (2004) 104-108.

Jo

[10] M.M. Islam, S.R. Chowdhury, G.S. Kumar, Spectroscopic and calorimetric studies on the binding of alkaloids berberine, palmatine and coralyne to double Stranded RNA polynucleotides, J. Phys. Chem. B 113 (2009) 1210-1224. [11] R. Sinha, M.M. Islam, K. Bhadra, G.S. Kumar, A. Banerjee, M. Maiti, The binding of DNA intercalating and non-intercalating compounds to A-form and protonated form of poly(rC).poly(rG): Spectroscopic and viscometric study, Bioorg. Med.Chem.14 (2006) 800-814. [12] A.A. Ghazaryan, Y.B. Dalyan, S.G. Haroutiunian, A. Tikhomirova, N. Taulier, J.W. Wells, T.V. Chalikian, Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes, J. Am. Chem. Soc. 128 (2006) 1914-1921. [13] R. Sinha, M. Hossain, G.S. Kumar, Interaction of small molecules with double-stranded RNA: Spectroscopic, viscometric, and calorimetric study of Hoechst and Proflavine binding to PolyCG structures, DNA Cell Biol. 28 (2009) 209-219. [14] A. Das, G.S. Kumar, Probing the binding of two sugar bearing anticancer agents aristololactam--D-glucoside and daunomycin to double stranded RNA polynucleotides: a combined spectroscopic and calorimetric study, Mol. BioSyst. 8 (2012) 1958-1969. 22

Journal Pre-proof [15] B.N. Briggs, A.J. Gaier, P.E. Fanwick, D.K. Dogutan, D.R. McMillin, Cationic copper(II) porphyrins intercalate into domains of double-stranded RNA, Biochemistry 51 (2012) 7496-7505. [16] A.B. Pradhan, H.K. Mondal, L. Haque, S. Bhuiya, S. Das, An overview on the interaction of phenazinium dye phenosafranine to RNA triple and double helices, Int. J. Biol. Macromol. 86 (2016) 345-351. [17] B. Saha, G.S. Kumar, Spectroscopic and calorimetric investigations on the binding of phenazinium dyes safranine-O and phenosafranine to double stranded RNA polynucleotides, J. Photochem. Photobiol. B 161 (2016) 129-140.

of

[18] A.Y. Khan, G.S. Kumar. Spectroscopic studies on the binding interaction of phenothiazinium dyes azure A and azure B to double stranded RNA polynucleotides. Spectrochim. Acta Mol. Biomol. Spectrosc. 152 (2016) 417–425.

ro

[19] P. Bhattacharjee, S. Sarkar, P. Pandya, K. Bhadra, Targeting different RNA motifs by beta carboline alkaloid, harmalol: a comparative photophysical, calorimetric, and molecular docking approach, J. Biomol. Struct. Dyn. 34 (2016) 2722-2740.

-p

[20] B. Krust, C. Callebaut, A.G. Hovanessian, Inhibition of entry of HIV into cells by poly(A).poly(U), AIDS Res. Hum. Retroviruses 9 (1993) 1087-1090.

lP

re

[21] B.K. Lee, Y.J. Yu, J.K. Youn, Effect of polyadenylic.polyuridylic acid on the proliferative responsiveness of mouse thymus and spleen cells, Yonsei Med. J. 31 (1990) 174181.

na

[22] H. Kato, O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. Uematsu, A. Jung, T. Kawai, K.J. Ishii, O. Yamaguchi, K. Otsu, T. Tsujimura, C-S. Koh, C.R. Sousa, Y. Matsuura, T. Fujita, S. Akira, Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses, Nature 441 (2006) 101-105.

Jo

ur

[23] K. Yan, L. Cheng, P. Liu, Z. Liu, S. Zhao, W. Zhu, Q. Wang, H. Wu, D. Han, Polyinosinic-Polycytidylic Acid perturbs ovarian functions through Toll-Like receptor 3mediated tumor necrosis factor a production in female mice biology of reproduction, Biol. Reprod. 93 (2015) 1-9. [24] A.A. Smorodintsev, O.A. Aksenov, I.K. Konstantinova, L.M. Vil'ner, A.V. Tinufanov, Comparative study of the toxicity of poly G-poly C and poly I-poly C in different objects, Vopr. Virusol. 2 (1978) 201-206. [25] M. Alda, M. McKinnon, R. Blagdon, J. Garnham, S. MacLellan, C. O'Donovan, T. Hajek, C. Nair, S. Dursun, G. MacQueen, Methylene blue treatment for residual symptoms of bipolar disorder: randomised crossover study, Br. J. Psychiatry. 210 (2017) 54-60. [26] T.C. Dumbarton, S.K. Gorman, S. Minor, O. Loubani, F. White, R. Green, Local cutaneous necrosis secondary to a prolonged peripheral infusion of methylene blue in vasodilatory shock, Ann. Pharmacother. 46 (2012) e6. [27] L. Vutskits, A. Briner, P. Klauser, E. Gascon, A.G. Dayer, J.Z. Kiss, D. Muller, M.J. Licker, D.R. Morel, Adverse effects of methylene blue on the central nervous system, Anesthesiology 108 (2008) 684-692.

23

Journal Pre-proof [28] S. Sohrabnezhada, A. Pourahmad, Comparison absorption of new methylene blue dye in zeolite and nanocrystal zeolite, Desalination 256 (2010) 84-89. [29] A. Kabir, G.S. Kumar, Targeting double-stranded RNA with spermine, 1-naphthylacetyl spermine and spermidine: a comparative biophysical investigation, J. Phys. Chem. B 118 (2014) 11050-11064. [30] J.D. McGhee, P.H.V. Hippel, Theoretical aspects of DNA-protein interactions: Cooperative and noncooperative binding of large ligands to a one dimensional homogeneous lattice, J. Mol. Biol. 86 (1974) 469-489. [31] H. Benesi, J. Hildebrand, A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703-2707.

of

[32] P. Job, Formation and stability of inorganic complexes in solution, Ann. Chim. 9 (1928) 113-203.

-p

ro

[33] P. Giri, G.S. Kumar, Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules, Arch. Biochem. Biophys. 474 (2008) 183-192.

re

[34] M.M. Islam, R. Sinha, G. S. Kumar, RNA binding small molecules: studies on t-RNA binding by cytotoxic alkaloids berberine, palmatine and the comparison to ethidium, Biophys. Chem. 125 (2007) 508-520.

lP

[35] K. Bhadra, M. Maiti, G. S. Kumar, Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine, Biochim. Biophys. Acta 1770 (2007) 1071-1080.

na

[36] W. Kohn, L.J. Sham, Self-consistent equations including exchange and correlation effects, Phys. Rev. 140 (1965) A1133-1138.

ur

[37] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys. 98 (1993) 5648-5652.

Jo

[38] C.T. Lee, W.T. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785-789. [39] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The Protein Data Bank, Nucleic Acids Res. 28 (2000) 235-242. [40] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a lamarckian genetic algorithm and an empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639-1662. [41] P. Paul, S.S. Mati, S.C. Bhattacharya, G.S. Kumar, Spectroscopic, calorimetric, cyclic voltammetric and molecular modeling studies of new methylene blue-polyadenylic acid interaction and comparison to thionine and toluidine blue O: Understanding self structure formation by planar dyes, Dyes Pigments 136 (2017) 205-218. [42] Y. Gilad, H. Senderowitz, Docking studies on DNA intercalators, J. Chem. Inf. Model. 54 (2014) 96-107.

24

Journal Pre-proof [43] C. G. Ricci, P. A. Netz, Docking studies on DNA-ligand interactions: building and application of a protocol to identify the binding mode, J. Chem. Inf. Model. 49 (2009) 19251935. [44] H.J. Lozano, B. García, N. Busto, J.M. Leal, Interaction of Thionine with Triple‑, Double‑, and Single-Stranded RNAs, J. Phys. Chem. B 117 (2013) 38-48. [45] N. Shahabadi, S. Hadidi, Spectroscopic studies on the interaction of calf thymus DNA with the drug Levetiracetam, Spectrochim Acta Mol. Biomol. Spectrosc. 96 (2012) 278-283. [46] R.H. Elmore, R.M. Wadkins, D.E. Graves, Cooperative binding of m-AMSA to nucleic acids, Nucleic Acids Res. 16 (1988) 9707-9719.

of

[47] N. Shahabadi, M. Maghsudi, Multi-spectroscopic and molecular modeling studies on the interaction of antihypertensive drug; methyldopa with calf thymus DNA, Mol BioSyst. 10 (2014) 338-347.

-p

ro

[48] D.V. Berdnikova, N.I. Sosnin, O.A. Fedorova, H. Ihmels, Governing the DNA-binding mode of styryl dyes by the length of their alkyl substituents - from intercalation to major groove binding, Org. Biomol. Chem. 16 (2018) 545-554.

re

[49] S. Agarwal, D.K. Jangir, R. Mehrotra, N. Lohani, M.R. Rajeswari, A structural insight into major groove directed binding of Nitrosourea derivative nimustine with DNA: A spectroscopic study, PLOS ONE 9 (2014) e104115.

Jo

ur

na

lP

[50] D. A. Lightner and J. Y. An, Circular dichroism of bilirubin amine heteroassociation complexes, Tetrahedron, 43 (1987) 4287-4296.

25

Journal Pre-proof FIGURE CAPTIONS Figure 1. Chemical structure of (a) methylene blue and (b) new methylene blue. Figure 2. Representative absorption spectral changes of (a) MB (4 M) treated with 0, 4, 8, 16, 32, 40, 48 M (curves 1-7) of poly(A).poly(U) and (b) NMB (3.5 M) treated with 0, 3.5, 7, 14, 28, 35, 42, 49 M (curves 1-8) of poly(A).poly(U). Experiments were performed at (201) o C in 20 mM sodium-cacodylate buffer at pH 7.2.

of

Inset: Scatchard plots for the binding of (a) MB (■) and (b) NMB (●) to poly(A).poly(U).

ro

Figure 3. Representative steady state fluorescence emission spectral changes of (a) MB (2 M)

-p

treated with 0, 2, 6, 14, 24, 30, 36 M (curves 1-7) of poly(A).poly(U) and (b) NMB (2 M)

re

treated with 0, 2, 6, 14, 24, 30, 36 M (curves 1-7) of poly(A).poly(U). All experiments were

lP

done in 20 mM sodium cacodylate buffer of pH 7.2.

Inset: Scatchard plots for the binding of (a) MB (■) and (b) NMB (●) to poly(A).poly(U).

na

Figure 4. Circular dichroic spectra of poly(A).poly(U) (60 M) treated with (a) 0, 6, 18, 24, 36,

ur

48, 60 M of MB (curves 1-7), (d) 0, 6, 18, 24, 36, 48, 60 M of NMB (curves 1-7); poly(C).poly(G) (60 M) treated with (b) 0, 6, 18, 24, 36, 48, 60 M of MB (curves 1-7), (e) 0,

Jo

6, 18, 24, 36, 48, 60 M of NMB (curves 1-7); poly(I).poly(C) (60 M) treated with (c) 0, 6, 18, 24, 36, 48, 60 M of MB (curves 1-7), (f) 0, 6, 18, 24, 36, 48, 60 M of NMB (curves 1-7). The expressed molar ellipticity () values are based on RNA concentration. Figure 5. Atomic force microscopy (AFM) images: (a) image of free poly(A).poly(U) within a scan area of 400 nm × 400 nm, (a) 3D image of poly(A).poly(U), (b) image of MBpoly(A).poly(U) complexes within a scan area of 550 nm × 550 nm, (b) 3D image of MBpoly(A).poly(U) complexes, (c) image of NMB-poly(A).poly(U) complexes within a scan area of 300 nm × 300 nm, (c) 3D image of NMB-poly(A).poly(U) complexes, (d) image of free 26

Journal Pre-proof poly(C).poly(G) within a scan area of 650 nm × 650 nm, (d) 3D image of poly(C).poly(G), (e) image of MB-poly(C).poly(G) complexes within a scan area of 1 m × 1 m, (e) 3D image of MB-poly(C).poly(G) complexes, (f) image of NMB-poly(C).poly(G) complexes within a scan area of 1.45 m × 1.45 m, (f) 3D image of NMB- poly(C).poly(G) complexes, (g) image of free poly(I).poly(C) within a scan area of 550 nm × 550 nm, (g) 3D image of poly(I).poly(C),

of

(h) image of MB- poly(I).poly(C) complexes within a scan area of 2.22 m × 2.22 m, (h) 3D image of MB- poly(I).poly(C) complexes, (i) image of NMB-poly(I).poly(C) complexes within a

ro

scan area of 1.3 m × 1.3 m, (i) 3D image of NMB- poly(I).poly(C) complexes.

-p

Figure 6. Energy minimized docked pose and closer view of (a) MB and (b) NMB in

re

poly(A).poly(U) showing intercalation mode of insertion between the base pairs. (c) and (d)

lP

represents same for poly(C).poly(G) showing partial intercalation inside the base pair. (e) and (f)

Jo

ur

na

represents the groove binding of MB and NMB respective in the minor groove of poly(I).poly(C)

27

Journal Pre-proof

Table 1: Binding parameters for the complexation of the two dyes with dsRNA evaluated from Scatchard analysis of the absorbance and fluorescence titration data a. Absorbance Dyes used MB

NMB

Fluorescence

RNA

K10-5 (M -1 )b

n

ω

Ki 10-5 (M -1 )

poly(A).poly(U)

0.105 ±0.36

2.08

87.4

9.17 ±0.36

poly(C).poly(G)

5.99±0.24

2.45

NA

5.99±0.24

poly(I).poly(C)

2.51±0.16

2.91

NA

2.51±0.16

poly(A).poly(U)

0.104 ±0.24

2.16

74.49

poly(C).poly(G)

nd

nd

poly(I).poly(C)

nd

nd

l a

n r u

a

nd nd

f o

n

ω

Ki 10-5 (M -1 )

0.114 ±0.17

2.12

78

8.89 ±0.17

5.81±0.32

2.52

NA

5.81±0.32

2.48±0.11

3.08

NA

2.48±0.11

7.71±0.24

0.084 ±0.43

2.22

84

7.05 ±0.43

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

o r p

e

r P

K10-5 (M -1 )b

Average of four determinations. b Binding constants (K) and the number of binding sites (n) refer to solution conditions of 20 mM cacodylate buffer, pH 7.2 at 20o C. ω is the cooperativity factor.

Jo

28

Journal Pre-proof Table 2: Binding parameters for the complexation of the dyes with RNA evaluated from BH plot of the absorbance and fluorescence titration dataa. Spectrophotometry Spectrofluorimetry RNA

MB KBH x10-5 M-1

NMB KBH x10-5 M-1

MB KBH x10-5 M-1

NMB KBH x10-5 M-1

9.14±0.16 5.64±0.07 2.33±0.11

7.42±0.15 4.93±0.08 1.81±0.14

Jo

ur

na

lP

re

-p

ro

of

poly(A).poly(U) 9.02±0.01 7.51±0.01 poly(C).poly(G) 5.72±0.09 4.87±0.06 poly(I).poly(C) 2.28±0.13 1.87±0.14 a The data presented are averages of three determinations.

29

Journal Pre-proof

Table 3: Energy value and ligand efficiency of MB and NMB molecules into the three polynucleotide bases. RNA

Binding energy (kJmol-1 )

Ligand efficiency

Intermolecular energy (kJmol-1 )

MB

poly(A).poly(U)

-30.1

-0.28

-35.9

poly(C).poly(G)

-26.8

-0.22

-30.6

poly(I).poly(C)

-24.4

-0.23

-29.5

poly(A).poly(U)

-27.5

-0.27

-30.9

poly(C).poly(G)

-26.4

-0.21

-26.0

poly(I).poly(C)

-23.3

-0.21

-26.8

ro

-p

re lP na ur Jo

NMB

of

Dyes used

30

Journal Pre-proof

Contributor Role P. Paul and G. S. Kumar conceived the idea and designed the experiments. G. S. Kumar supervised the experimental work. P. Paul performed the experiments, analyzed the data and prepared all figures. S. S. Mati performed the molecular modeling studies, analyzed the data, prepared related figure and wrote the portion related to modeling. P. Paul and G. S. Kumar wrote the manuscript except the molecular modeling portion. All the authors discussed the data,

CRediT author statement Paul: Conceptualization,

Methodology,

Visualization,

S. S. Mati:

Investigation,

Analysis,

Software,

re

Writing- Original draft preparation

-p

P.

ro

of

provided critical feedback and contributed to the final manuscript.

Methodology, Software, Investigation, Writing some portions of original draft

lP

preparation

Jo

ur

na

G. S. Kumar: Supervision, Validation, Writing - Review & Editing

31

Journal Pre-proof Declaration of interests

Jo

ur

na

lP

re

-p

ro

of

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

32

Journal Pre-proof

Highlights  Higher binding of MB to dsRNA over the other dyes by intercalation was observed.  Perturbation of dsRNA conformation was observed on binding with the dye.  AFM imaging predicted alteration in RNA molecular structures.

Jo

ur

na

lP

re

-p

ro

of

 The probable binding location of the dyes was investigated from docking simulation.

33

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6