Role of hydroxyl groups in the B-ring of flavonoids in stabilization of the Hoogsteen paired third strand of Poly(U).Poly(A)*Poly(U) triplex.

We have reported the interaction of two flavonoids namely quercetin (Q) and morin (M) with double stranded poly(A).poly(U) (herein after A.U) and triple stranded poly(U).poly(A)*poly(U) (herein after U.A*U, dot represents the Watson-Crick and asterisk represents Hoogsteen base pairing respectively) in this article. It has been observed that relative positions of hydroxyl groups on the B-ring of the flavonoids affect the stabilization of RNA. The double strand as well as the triple strand of RNA-polymers become more stabilized in presence of Q, however both the duplex and triplex remain unaffected in presence of M. The presence of catechol moiety on the B-ring of Q is supposed to be responsible for the stabilization. Moreover, after exploiting a series of biophysical experiments, it has been found that, triple helical RNA becomes more stabilized over its parent duplex in presence of Q. Fluorescence quenching, viscosity measurement and helix melting results establish the fact that Q binds with both forms of RNA through the mode of intercalation while M does not bind at all to either forms of RNA.

Exploring the comparative binding aspects of benzophenanthridine plant alkaloid chelerythrine with RNA triple and double helices: a spectroscopic and calorimetric approach.

A comparative study on the interaction of a benzophenanthridine alkaloid chelerythrine (CHL) with RNA triplex poly(U).poly(A)*poly(U) (hereafter U.A*U, .(dot) and *(asterisk) represent Watson-Crick and Hoogsteen base pairing respectively) and its parent duplex poly(A).poly(U) (A.U) was carried out by using a combination of various spectroscopic, viscometric and calorimetric techniques. The interaction was characterized by hypochromic and bathochromic effects in the absorption spectrum, the increase of thermal melting temperature, enhancement in solution viscosity, and perturbation in the circular dichroic spectrum. The binding constant calculated by using spectrophotometric data was in the order of 10(5) for both forms of RNA, but it was greater for triplex RNA (30.2 × 10(5) M(-1)) than duplex RNA (3.6 × 10(5) M(-1)). Isothermal titration calorimetric data are in good agreement with the spectrophotometric data. The data indicated stronger binding of CHL to the triplex structure of RNA compared to the native duplex structure. Thermal melting studies indicated greater stabilization of the Hoogsteen base paired third strand of the RNA triplex compared to its Watson-Crick strands. The mode of binding of CHL to both U.A*U and A.U was intercalation as revealed from fluorescence quenching, viscosity measurements and sensitization of the fluorescence experiment. Thermodynamic data obtained from isothermal calorimetric measurements revealed that association was favoured by both a negative enthalpy change and a positive entropy change. Taken together, our results suggest that chelerythrine binds and stabilizes the RNA triplex more strongly than its respective parent duplex. The results presented here may be useful for formulating effective antigene strategies involving benzophenanthridine alkaloids and the RNA triplex.

Influence of position of hydroxyl group of flavonoids on their binding with single stranded polyriboadenylic acid: A spectroscopic evaluation.

Single stranded polyriboadenylic acid [poly (rA)] has been accepted widely as a suitable drug target owing to its vital role in the development of cancer since it controls gene expression during cell growth and differentiation. The biological properties of poly (rA) depend on its structural morphology. Pharmacologically active flavonoids can act as suitable binders to poly (rA) and significantly change its biophysical properties. Different factors favour flavonoid-poly (rA) binding. In our present work we have explored the role played by the position of hydroxyl groups in the flavonoids namely 3, 5, 6 and 7 hydroxyflavones in their course of interaction with poly (rA). A range of spectroscopic experiments reveal that 3HF binds best to poly (rA) among the four chosen flavonoids. This is probably due to the presence of a hydroxyl group in '3' position that enables it to exhibit ESIPT phenomenon which is missing for the other used flavonoids.

In-depth investigation of the binding of flavonoid taxifolin with bovine hemoglobin at physiological pH: Spectroscopic and molecular docking studies.

The use of bioactive flavonoids as drugs has long mesmerized the scientific world. Their small size and planar structure enables them to interact with limitless substrates especially biomolecules. Taxifolin is a flavonoid well known for its anti-oxidizing and metal chelating properties. Its interaction with a few biomolecules has been studied so far to exploit its pharmacological activities. Hemoglobin, an iron containing macromolecule acts as a major carrier protein and is also associated with the occurrence of many diseases. Our present study lays emphasis on the interaction of flavanonol taxifolin with bovine hemoglobin at physiological pH. This was achieved by monitoring the changes in the absorbance, fluorescence, anisotropic, lifetime and circular dichroic spectra. Benesi-Hildebrand plot determined a binding constant value of 20.0 × 103 M-1 at 25 °C. Stern-Volmer quenching studies reveal that the binding is associated with a static mode of quenching. The complexation is thermodynamically favored as indicated by the negative value of enthalpy and positive value of entropy changes seen from the van't Hoff plot. Theoretical DFT calculations were used to find out an optimized geometry and HOMO-LUMO energy gap for taxifolin. Molecular docking studies revealed the location of taxifolin inside the hemoglobin moiety.

Elucidation of the association of potential chemotherapeutic alkaloid chelerythrine with bovine hemoglobin by experimental probing and molecular docking simulation.

Chelerythrine (CHL) is a pharmacologically important molecule that appears in positively charged iminium and neutral alkanolamine form on varying the pH. Association of bovine hemoglobin (BHb) with iminium and alkanolamine forms of CHL is explored employing several spectroscopic and theoretical tools. Our results revealed that iminium form of CHL shows greater binding affinity than the neutral alkanolamine form, with nearly one binding site on the protein for both forms. Thermodynamic data showed that the iminium binding to BHb was characterized by negative enthalpy and positive entropy changes while the association of the alkanolamine CHL was accompanied with both positive enthalpy and entropy changes. Both forms of CHL have been found to quench the intrinsic fluorescence of BHb. From Förster's resonance energy transfer (FRET) studies, the binding distance between the energy acceptor (CHL) and donor (ß-Trp 37 of BHb) was found to be optimum for fluorescence quenching to occur. The conformational transformation of BHb induced by CHL complexation showed greater unfolding of the protein architecture for the iminium interaction from CD spectroscopy. Molecular docking study revealed that both iminium and alkanolamine form of CHL reside near ß-Trp 37 at the α1ß2 interface of BHb.

Self-structure assembly in single stranded polyriboadenylic acid by benzophenanthridine alkaloid: Spectroscopic and calorimetric exploration.

Present study allows us a better understanding of the interaction of nitidine, a benzophenanthridine alkaloid with single stranded polyriboadenylic acid [ss-poly (rA)]. The interaction leads to self-structure induction in ss-poly (rA) under the experimental condition of pH 7.0. Interaction of nitidine with ss-poly (rA) was ascertained by monitoring the change in absorbance, fluorescence intensity and circular dichroism values. Binding mode of nitidine with ss-poly (rA) was observed to be intercalation as confirmed from the quenching and viscometric studies. The association was characterized by both negative enthalpy and entropy changes accompanying with a moderately high binding constant of 5.10×105M-1. Nitidine induced double helical organization in single stranded poly (rA) under the experimental pH.

Spectroscopic, photophysical and theoretical insight into the chelation properties of fisetin with copper (II) in aqueous buffered solutions for calf thymus DNA binding.

Fisetin (FTN) and its metal chelates are critically important since this bioflavonoid possesses wide range of pharmacological properties. Usually, metal binding property enhances the pharmaceutical activity of FTN. Thus in this report, we investigated the complexation of FTN with biologically essential metal ion Cu2+ and further examined the effect of such complexation on calf thymus DNA (CT DNA) binding in comparison with free FTN. We have characterized the complex formation of FTN with Cu2+ using UV-visible, fluorimetric and FTIR studies. Within our experimental concentration range we found that, FTN forms a 2:1 complex with Cu2+ in terms of FTN:Cu2+. Spectroscopic analysis revealed that both FTN and FTN2-Cu2+ complex bind with CT DNA and the binding constant is higher for free FTN. Perturbation of circular dichroism spectrum of CT DNA was observed in presence of free FTN due to structural alteration in DNA double helix. Viscometric, thermal melting and fluorescence quenching study confirm that FTN intercalates in between the base pairs of CT DNA while its Cu (II) complex acts as a groove binder. Molecular docking study further confirms that FTN intercalates into AT rich region of CT DNA while its Cu (II) complex binds at the minor groove.

Structural alteration of low pH, low temperature induced protonated form of DNA to the canonical form by the benzophenanthridine alkaloid nitidine: Spectroscopic exploration.

Polymorphism of DNA plays a very important part of research relating to the drug-DNA interactions. Here main focus of our investigation is to monitor the interaction of the benzophenanthridine plant alkaloid, nitidine (NIT) with two different forms of DNA i.e. B-DNA and protonated form of DNA maintaining proper temperatures and buffer conditions. Binding interaction of NIT was ascertained from the UV-Visible spectroscopic and spectrofluorimetric titration experiments. Binding constants of the interactions of NIT with different polymorphic forms were calculated from UV-absorption study. The binding constants were 3.8 × 105 M-1 and 1.3 × 105 M-1 for B-DNA and protonated DNA respectively. Red shift in the absorption maxima of NIT on binding with DNA, comparatively greater relative quenching of fluorescence intensity of free NIT than bound NIT, perturbation in the CD spectrum of DNA in presence of NIT confirmed the mode of binding as intercalation. Moreover, spectropolarimetric experiment confirms that left handed protonated form of DNA gets partially converted to the canonical B form of DNA while binds with NIT. Besides the CD experiment, thermal melting experiment also showed that on binding with NIT stabilization of protonated DNA was increased to an appreciable extent.

Multispectroscopic and Theoretical Exploration of the Comparative Binding Aspects of Bioflavonoid Fisetin with Triple- and Double-Helical Forms of RNA.

The interactions of RNA triplex (U.A*U) and duplex (A.U) with naturally occurring flavonoid fisetin (FTN) have been examined at pH 7.0 using various spectroscopic, viscometric, and theoretical studies. Experimental observations showed that the ligand binds with both double- and triple-helical forms of RNA, although the binding affinity is greater for the triplex structure (5.94 × 106 M-1) compared to that for the duplex counterpart (1.0 × 105 M-1). Thermal melting experiments revealed that the Hoogsteen base-paired third strand of triplex was stabilized to a greater extent (∼14 °C) compared with the Watson-Crick base-paired second strand (∼4 °C) in the presence of FTN. From fluorimetric study, we observed that U.A*U and A.U primarily bind to the photoproduced tautomer of FTN in the excited state. Steady-state and time-resolved anisotropy measurements illustrate considerable modulations of the spectroscopic properties of the tautomeric FTN within the RNA environment. Viscometric, fluorescence quenching, and thermal melting studies all together support the mode of binding to be intercalation. Theoretical study explains the experimental absorption and emission (dual fluorescence) behavior of FTN along with the excited-state intramolecular proton transfer process.

Third strand stabilization of poly(U)·poly(A)* poly(U) triplex by the naturally occurring flavone luteolin: A multi-spectroscopic approach.

Naturally occurring flavonoid luteolin (LTN) was found to interact with double stranded poly(A).poly(U) and triple stranded poly(U)·poly(A)*poly(U) with association constants of the order of 104M-1. The association was monitored by various spectroscopic and viscometric techniques. Non-cooperative binding was observed for the association of LTN with two different polymorphic forms of RNA. Intercalation mode of binding was confirmed by fluorescence quenching and viscometric experiments. Thermal melting profiles indicated greater stabilization of the Hoogsteen base paired third strand (∼16°C) compared to Watson-Crick double strand (∼5°C) of RNA by LTN. Since the interaction of naturally occurring small molecules with RNA is an active area of research, this study has led to great openings to explore LTN as RNA targeted therapeutic agent.

Spectroscopic study on the binding of chelerythrine with duplex poly (rA): A model of RNA intercalation.

Here we have reported a detail study on the interaction of the benzophenanthridine alkaloid chelerythrine (CHL) with double stranded polyriboadenylic acid [ds poly (rA)] by exploiting various spectroscopic techniques. The alkaloid shows high binding affinity (binding constant is 1.10×105M-1) towards the double stranded RNA as revealed from Scatchard plot. The binding was confirmed by hypochromic effect in the UV-vis spectrum of CHL, increase in fluorescence intensity of CHL and perturbations of the circular dichroism (CD) spectrum of ds poly (rA). Later fluorescence quenching, cooperative CD melting transition, viscometric and molecular modeling studies establish the fact that the alkaloid binds to the ds poly (rA) by the mechanism of intercalation. Thermodynamic parameters obtained from the isothermal titration calorimetric (ITC) study show that the binding is favoured by negative enthalpy and small positive entropy changes. This report may be a model for intercalation of small molecule like CHL to the double stranded RNA.

Inhibitory effects of the dietary flavonoid quercetin on the enzyme activity of zinc(II)-dependent yeast alcohol dehydrogenase: Spectroscopic and molecular docking studies.

A multispectroscopic exploration was employed to investigate the interaction between the metallo-enzyme alcohol dehydrogenase (ADH) from yeast with bioflavonoid quercetin (QTN). Here, we have characterized the complex formation between QTN and Zn2+ in aqueous solution and then examined the effect of such complex formation on the enzymatic activity of a zinc(II)-dependent enzyme alcohol dehydrogenase from yeast. We have observed an inhibition of enzymatic activity of ADH in presence of QTN. Enzyme inhibition kinetic experiments revealed QTN as a non-competitive inhibitor of yeast ADH. Perturbation of Circular dichroic (CD) spectrum of ADH in presence of QTN is observed due to the structural changes of ADH on complexation with the above flavonoid. Our results indicate a conformational change of ADH due to removal of Zn2+ present in the enzyme by QTN. This was further established by molecular modeling study which shows that the flavonoid binds to the Zn2+ ion which maintains the tertiary structure of the metallo-enzyme. So, QTN abstracts only half of the Zn2+ ions present in the enzyme i.e. one Zn2+ ion per monomer. From the present study, the structural alteration and loss of enzymatic activity of ADH are attributed to the complex formation between QTN and Zn2+.

Micelle assisted structural conversion with fluorescence modulation of benzophenanthridine alkaloids.

In this study we have reported the anionic surfactant (Sodium dodecyl sulfate, SDS) driven structural conversion of two benzophenanthridine plant alkaloids namely Chelerythrine (herein after CHL) and Sanguinarine (herein after SANG). Both the alkaloids exist in two forms: the charged iminium and the neutral alkanolamine form. The iminium form is stable at low pH (<6.5) and the alkanolamine form exists at higher pH (>10.1). The fluorescence intensity of the alkanolamine form is much stronger than the iminium form. The iminium form of both the alkaloids remains stable whereas the alkanolamine form gets converted to the iminium form in the SDS micelle environment. The iminium form possesses positive charge and it seems that electrostatic interaction between the positively charged iminium and negatively charged surfactant leads to the stabilization of the iminium form in the Stern layer of the anionic micelle. Whereas the conversion of the alkanolamine form into the iminium form takes place and that can be monitored in naked eye since the iminium form is orange in colour and the alkanolamine form has blue violet emission. Such a detail insight about the photophysical properties of the benzophenanthridine alkaloids would be a valuable addition in the field of alkaloid-surfactant interaction.

Molecular Aspects of the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin.

The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and isothermal titration calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time-resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA subdomain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor), respectively. Circular dichroism (CD) study demonstrated that the α-helical organization of the protein is reduced due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favored by negative enthalpy change and positive entropy change.

An overview on the interaction of phenazinium dye phenosafranine to RNA triple and double helices.

Triple helical nucleic acids or triplex are formed from the combination of three oligonucleotides. In the double stranded form the base pairs are joined through Watson-Crick base pairing while the third strand of the triplex is joined through Hoogsteen base pairing. The Hoogsteen base paired strands are less stable compare to the Watson-Crick strands. Thus stabilization of the Hoogsteen strand gains importance due to the implication of stable triplexes in various biological processes. For this reason here we have monitored the effect of phenosafranine, a phenazinium dye on the structure of poly(U).poly(A)*poly(U) triplex. Our data revealed that the dye has higher binding affinity to the RNA triplex (K'=3.7 × 10(5) M(-1)) compared to the parent duplex (K'=1.9 × 10(5) M(-1)) form. Through a series of spectroscopic and viscometric study we have found that the dye binds to triplex or duplex through the mechanism of intercalation and it stabilizes the Watson-Crick strand while the Hoogsteen strand remains unaffected.

Deciphering the Positional Influence of the Hydroxyl Group in the Cinnamoyl Part of 3-Hydroxy Flavonoids for Structural Modification and Their Interaction with the Protonated and B Form of Calf Thymus DNA Using Spectroscopic and Molecular Modeling Studies.

Studies on the interaction of naturally occurring flavonoids with different polymorphic forms of nucleic acid are helpful for understanding the molecular aspects of binding mode and providing direction for the use and design of new efficient therapeutic agents. However, much less information is available on the interactions of these compounds with different polymorphic forms of DNA at the molecular level. In this report we investigated the interaction of two widely abundant dietary flavonoids quercetin (Q) and morin (M) with calf thymus (CT) DNA. Spectrophotometric, spectropolarimetric, viscosity measurement, and molecular docking simulation methods are used as tools to delineate the binding mode and probable location of the flavonoids and their effects on the stability and conformation of DNA. It is observed that in the presence of the protonated form of DNA the dual fluorescence of Q and M resulting from the excited-state intramolecular proton transfer (ESIPT) is modified significantly. Structural analysis showed Q and M binds weakly to the B form (groove binding) compared to the protonated form of CT DNA (electrostatic interaction). In both cases, Q binds strongly to both forms of DNA compared to M.

Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.