Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules

Self-structure induction in single stranded poly(A) has been one typical example of the various ways that could be used to modulate nucleic acid structural aspects through binding of small molecules. For the first time, the interaction between a series of small molecules and poly(A) has been investigated to understand the nature of the structural features in DNA binding small molecules that could be responsible for the formation of self-structure in single stranded poly(A) molecules. Classical intercalators like ethidium, coralyne, quinacrine and proflavine, partial intercalators like berberine and palmatine and classical minor groove binders like hoechst 33258 and DAPI have been chosen for this study. The binding of each of these molecules to poly(A) has been characterized by absorption spectral titration, job plot and isothermal titration calorimetry. Self-structure formation was monitored from circular dichroic melting, optical melting and differential scanning calorimetry. The results revealed that while all the intercalators studied induced self-structure formation, partial intercalators did not induce the same in poly(A). Of the two classical DNA minor groove binding molecules investigated, hoechst was effective in inducing self-structure while DAPI was ineffective. Self-structure induction in poly(A) was observed to be directly linked to the cooperative binding of the molecules to poly(A) in that all the molecules that bound cooperatively induced self-structure in poly(A). Structural and thermodynamic aspects of the interaction leading to self-structure formation are described.     

Interaction of aristololactam-β-D-glucoside and daunomycin with poly(A): spectroscopic and calorimetric studies

The binding of two sugar containing antibiotics viz. aristololactam-β-D-glucoside and daunomycin with single and double stranded poly(A) was investigated by spectroscopic and calorimetric studies. The binding affinity of daunomycin to ss poly(A) was of the order of 10⁶ M^− 1 and that to ds poly(A) was of the order of 10⁵ M^− 1. Aristololactam-β-D-glucoside showed a relatively weaker binding with an affinity of the order of 10⁴ M^− 1 with both the conformations of poly(A). Fluorescence studies showed maximum quenching for daunomycin-ss poly(A) complexes. The binding constants calculated from fluorescence spectroscopy were in good agreement with that obtained from UV spectroscopy. Moderate perturbation of circular dichroic spectra of both the conformations of poly(A) in presence of these molecules with concomitant formation of prominent extrinsic CD bands in the 300-450 nm region further revealed the association. Isothermal titration calorimetry results showed an overall entropy driven binding in all the four systems though the entropy change was maximum in daunomycin-ss poly(A) binding. The binding affinity was also maximum for daunomycin-ss poly(A) and varied as daunomycin-ds poly(A)>aristololactam-β-D-glucoside-ds poly(A)>aristololactam-β-D-glucoside-ss poly(A). A 1:1 binding stoichiometry was observed in all the cases, as confirmed by Job plot analysis, indicating the interaction to consist of a single binding mode. Ferrocyanide quenching studies showed good stacking interaction in all cases but was best for daunomycin-ss poly(A) interaction. No self-structure formation was observed in poly(A) with both daunomycin and aristololactam-β-D-glucoside suggesting the hindrance of the sugar moiety for such structural organization.     

Small molecule–RNA interaction: Spectroscopic and calorimetric studies on the binding by the cytotoxic protoberberine alkaloid coralyne to single stranded polyribonucleotides

Background

RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of the alkaloid coralyne to poly(G), poly(C), poly(I) and poly(U) in the light of its ability in inducing self-structure in poly(A).

Methods

Multifaceted experimental techniques like competition dialysis, absorption, fluorescence, circular dichroism and calorimetry were employed. Salt dependence and temperature dependence of the binding was also elucidated.

Results

Results of competition dialysis, absorption and fluorescence studies revealed that coralyne binds strongly to the polypurines, poly(G) and poly(I) compared to the polypyrimidines, poly(U) and poly(C). Partial intercalative binding due to the stacking of the molecules between the bases was envisaged. The binding was predominantly enthalpy driven with favourable entropy term with a large favourable non-electrostatic contribution revealed from salt dependent data and the dissection of the free energy. The heat capacity change of − 125 and − 119 cal/mol K^− 1 respectively for poly(G) and poly(I) and the partial enthalpy–entropy compensation phenomenon observed confirmed the involvement of multiple weak noncovalent interactions. Circular dichroism studies provided evidence for significant perturbation of the conformation of the RNAs, but no self-structure induction was evident in any of the polymers under the condition of the study.

Conclusions

This study presents a complete structural and thermodynamic profile of coralyne interaction to four single stranded RNA polymers.

General significance

The study for the first time elucidates the base specificity of coralyne–RNA complexation at the single stranded level.     

Berberine-DNA complexation: new insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K(a) versus [Na(+)] for poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (DeltaCp(o)) of berberine binding to poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), Clostridium perfringens and calf thymus DNA were -98, -140, -120 and -110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

Molecular aspects on the specific interaction of cytotoxic plant alkaloid palmatine to poly(A)

The interaction of the protoberberine alkaloid palmatine with single and double stranded structures of poly(A) was studied by various biophysical techniques. Comparative binding studies were also performed with double stranded DNA, t-RNA, poly(C)·poly(G), poly(U) and poly(C). The results of competition dialysis, fluorescence, and absorption spectral studies converge to reveal the molecular aspects of the strong and specific binding of palmatine to single stranded poly(A). The binding affinity of palmatine to natural DNA, t-RNA and double stranded poly(A) was weaker while no binding was apparent with single stranded poly(U), poly(C) and double stranded poly(C)·poly(G). The strong affinity of the alkaloid to single stranded poly(A) in comparison to the double stranded structure was also revealed from circular dichroic and viscometric studies. The effect of [Na⁺] ion concentration on the binding process revealed the significant role of electrostatic forces in the complexation. The presence of bound alkaloid also remarkably affected denaturation–renaturation of stacked helical poly(A). The energetics of the strong binding to poly(A) was studied from thermodynamic estimation from van Hoff’ analysis of the temperature dependent binding constants and ultra sensitive isothermal titration calorimertry, both suggesting the binding to be exothermic and enthalpy driven. This study provides detailed insight into the binding specificity of the natural alkaloid to single stranded poly(A) over several other single and double stranded nucleic acid structures suggesting its potential as a lead compound for RNA based drug targeting.     

Interaction of isoquinoline alkaloid palmatine with deoxyribonucleic acids: binding heterogeneity, and conformational and thermodynamic aspects

The binding heterogeneity, conformational aspects, and energetics of the interaction of the cytotoxic plant alkaloid palmatine have been studied with various natural and synthetic DNAs. The alkaloid binds to calf thymus and Escherichia coli DNA that have mixed AT and GC sequences in almost equal proportions with positive cooperativity, while, with Clostridium perfringens and Micrococcus lysodeikticus DNA with predominantly high AT and GC sequences, respectively, noncooperative binding was observed. On further investigation with synthetic DNAs, the binding was observed to be cooperative with polymers like poly(dA).poly(dT) and poly(dG).poly(dC) having poly(purine)poly(pyrimidine) sequences, while with polymers poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT) and poly(dG-dC).poly(dG-dC), which have alternating purine-pyrimidine sequences, a non-cooperative binding phenomenon was observed. This suggests the binding heterogeneity of palmatine to the two types of sequences of base pairs. Circular dichroism (CD) studies revealed that the binding induced conformational changes in all the DNAs, but more importantly, the bound alkaloid molecules acquired induced optical activity, and the extent was dependent on the AT content and showed AT base-pair specificity. Energetics of the interaction of the alkaloid studied by highly sensitive isothermal titration calorimetry revealed that the binding was in most cases exothermic and favored by both enthalpy and entropy changes, while, in the case of the homo and hetero AT polymers, the same was predominantly entropy-driven. This study defines base-pair-dependent heterogeneity, conformational aspects, and energetics of palmatine binding to DNA.     

Probing the binding of two sugar bearing anticancer agents aristololactam-β-(D)-glucoside and daunomycin to double stranded RNA polynucleotides: a combined spectroscopic and calorimetric study

The plant alkaloid aristololactam-β-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-β-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-β-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.     

Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study

Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.     

RNA specific molecules: cytotoxic plant alkaloid palmatine binds strongly to poly(A)

The cytotoxic plant alkaloid palmatine was found to bind strongly by partial intercalation to single stranded poly(A) structure with binding affinity (Ka) of (8.36+/-0.26) x 10(5) M(-1). The binding of palmatine was characterized to be exothermic and enthalpy driven with one palmatine for every two adenine residues. On the other hand, the binding to the double stranded poly(A) has been found to be significantly weak. This study identifies poly(A) as a potential bio-target for the alkaloid palmatine and its use as a lead compound in antitumor drug screening.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K_a versus [Na⁺] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp^o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were − 98, − 140, − 120 and − 110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

The effect of Cephalotaxus group alkaloids on elongation of the polypeptide chain in human ribosomes

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.     

Poly(8-bromodeoxyadenylic acid): properties of the polymer and contrast with the ribopolynucleotide analogue

Introduction of the bulky 8-bromo substituent into adenine residues of polynucleotides has strikingly different consequences in the deoxy- and ribopolynucleotide series. Poly(r8BrA) was found in earlier studies to form a very stable double-helical self-structure but not to undergo interaction with potentially complementary polynucleotides. We find that poly(d8BrA), in contrast, does not form an ordered self-structure in 0.1 M Na+ but appears to exist as an electrostatically expanded rigid rod with unusual circular dichroism (CD) properties at very low ionic strength. The deoxy polymer, moreover, readily forms double helices with either deoxy or ribo pyrimidine polynucleotides, studied by UV, CD, and IR spectroscopy. These complexes are destabilized, relative to those formed by poly(dA), possibly because energy is needed to convert the purine residues from a more stable syn to an anti conformation, required for heteroduplex formation. The CD spectrum of (d8BrA)n X (dT)n is similar to that of B DNA. The deoxy-ribo hybrids (d8BrA)n X (rU)n and (d8BrA)n X (rBrU)n have CD spectra resembling those of A DNA or RNA. Unlike other deoxy-deoxy pairs (d8BrA)n X (dBrU)n, however, has a CD spectrum resembling RNA and other helices having the A form.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding

A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T_m ≈ 60°C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K_a >10⁷ M⁻¹), and that the crescent shape of coralyne appears necessary for poly(A) binding. We also show that the binding of similar ligands to poly(A) can be highly cooperative. For one particular ligand, at least six ligand molecules are required to stabilize the poly(A) self-structure at room temperature. This highly cooperative binding produces very sharp transitions between unstructured and structured poly(A) as a function of ligand concentration. Given the fact that junctions between Watson–Crick and A·A duplexes are tolerated, we propose that poly(A) sequence elements and appropriate ligands could be used to reversibly drive transitions in DNA and RNA-based molecular structures by simply diluting/concentrating a sample about the poly(A)-ligand ‘critical concentration’. The ligands described here may also find biological or medicinal applications, owing to the 3′-polyadenylation of mRNA in living cells.     

Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bond formation

The aim of the present study was to investigate homoharringtonine alkaloid effect on: (i) the nonenzymatic and eEF-1-dependent Phe-tRNAPhe binding to poly(U)-programmed human placenta 80 S ribosomes; (ii) diphenylalanine synthesis accompanying nonenzymatic Phe-tRNAPhe binding; and (iii) acetylphenylalanyl-puromycin formation. Neither nonenzymatic nor eEF-1-dependent Phe-tRNAPhe binding were noticeably affected by the alkaloid, whereas diphenylalanine synthesis and puromycin reaction were strongly inhibited by homoharringtonine. It has been proposed that the site of homoharringtonine binding on 80 S ribosomes should overlap or coincide with the acceptor site of the ribosome.     

Targeting different RNA motifs by beta carboline alkaloid,harmalol: a comparative photophysical,calorimetric, and molecular docking approach

RNA has attracted recent attention for its key role in gene expression and targeting by small molecules for therapeutic intervention. This work focuses towards understanding interaction of harmalol, a DNA intercalator, with RNAs of different motifs viz. single-stranded A-form poly(A), double-stranded A-form of poly(C)·poly(G), and clover leaf tRNA^phe by different spectroscopic, calorimetric, and molecular modeling techniques. Results of this study converge to suggest that (i) binding constant varied in the order poly(C)·poly(G)?>?tRNA^phe > poly(A), (ii) non-cooperative binding of harmalol to poly(C)·poly(G) and poly(A) and cooperative binding with tRNA^phe, (iii) significant structural changes of poly(C)·poly(G) and tRNA^phe with concomitant induction of optical activity in the bound achiral alkaloid molecules, while with poly(A) no induced Circular dichroism (CD) perturbation was observed, (iv) the binding was predominantly exothermic, enthalpy-driven, entropy-favored with poly(C)·poly(G), while it was entropy driven with tRNA^phe and poly(A), (v) a hydrophobic contribution and comparatively large role of non polyelectrolytic forces to Gibbs energy changes with poly(C)·poly(G) and tRNA^phe and (vi) intercalated state of harmalol inside poly(C)·poly(G) structure as revealed from molecular docking was supported by the viscometric and ferrocyanide quenching data. All these findings unequivocally pointed out that harmalol prefers binding with poly(C)·poly(G), compared to tRNA^phe and poly(A); this results serve as data for the development of RNA-based antiviral drugs.     

Molecular aspects on the interaction of aristololactam-beta-D-glucoside with H(L)-form deoxyribonucleic acid structures

Synthetic alternating GC-rich DNA polymers can adopt Hoogsteen base-paired structures (H(L)-form) under the influence of low pH and temperature. The interaction of aristololactam-beta-D-glucoside (ADG), a natural glucoside derivative of aristolochia group of alkaloids, with protonation-induced structures (H(L)-form) of poly(dG-dC).poly(dG-dC) and poly(dG-m(5)dC).poly(dG-m(5)dC) has been studied using different biophysical techniques. The binding of ADG to protonated DNA is characterized by typical hypochromism and bathochromism of the absorption spectrum of the alkaloid, quenching of steady state fluorescence intensity, decrease in quantum yield, increase in fluorescence polarization anisotropy values, increase in thermal transition temperature of polynucleotides following alkaloid binding and perturbation of circular dichroic spectrum of polynucleotides as a result of its interaction with the alkaloid. Scatchard analysis of the data indicates that ADG binds to protonated structures in a nonlinear noncooperative manner. The binding parameters determined from spectrophotometric titration data employing excluded site model indicate that protonated poly(dG-m(5)dC).poly(dG-m(5)dC) is more favorable for ADG binding than the corresponding nonmethylated analog. The binding of ADG to protonated structures renders a higher degree of stabilization against thermal denaturation compared to respective B-form-ADG interactions and induces a conformational switch to a bound altered form which is different from its interaction with B- and Z-form DNA structures. Thermodynamic parameters (Delta G degrees, Delta H degrees and Delta S degrees ) obtained by van't Hoff analysis of the data indicate that the binding of alkaloid to protonated structures is an exothermic process and the binding free energy arises primarily from a negative enthalpy change. Moreover, the binding leads to an increase in the contour length of protonated DNAs. These results suggest that ADG possibly binds to protonated DNAs by the mechanism of intercalation.