Interaction of isoquinoline alkaloids with an RNA triplex: structural and thermodynamic studies of berberine, palmatine, and coralyne binding to poly(U).poly(A)(*)poly(U).

The interaction of two natural protoberberine alkaloids berberine and palmatine and the synthetic derivative coralyne with the RNA triplex poly(U).poly(A)(*)poly(U) was studied using various biophysical and calorimetric techniques. All the three alkaloids bind noncooperatively to the triplex. The affinity of berberine and palmatine was in the order of 10(5) M(-1), while that of coralyne was one order higher as inferred from spectroscopic studies. The alkaloids stabilized the Hoogsteen base-paired third strand of the triplex without affecting the stability of the duplex. Fluorescence quenching and viscosity studies gave convincing evidence for the partial intercalation of berberine and palmatine and a true intercalative binding of coralyne to the triplex. This was further supported from the significant polarization of the emission spectra of the complex and the energy transfer from the base triplets to the alkaloids. Circular dichroic studies suggested that the conformation of the triplex was perturbed significantly by the binding of the alkaloids, being more by coralyne compared to berberine and palmatine and also evidenced by the generation of strong induced optical activity in the bound coralyne molecules. Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by a predominantly large negative enthalpy change (DeltaH degrees = -5.42 kcal/mol) with small favorable entropy contribution (TDeltaS degrees = 2.02 kcal/mol) in berberine, favored by almost equal negative enthalpy (DeltaH degrees = -3.93 kcal/mol) and entropy changes (TDeltaS degrees = 3.89 kcal/mol) in palmatine and driven by predominant entropy contributions (DeltaH degrees = -1.84 and TDeltaS degrees = 7.44 kcal/mol) in coralyne. These results advance our knowledge on the binding of small molecule isoquinoline alkaloids that are specific binders of RNA structures, particularly triplexes.

RNA targeting by DNA binding drugs: structural, conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H(L)-form of poly(rC).poly(rG).

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4',6-diamidino-2-phenylindole (DAPI) with the right handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form of poly(rC).poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H(L)-form of poly(rC).poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H(L)-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H(L)-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H(L)-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA-ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium.

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.

Interaction of small molecules with double-stranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to PolyCG structures.

Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

The binding of DNA intercalating and non-intercalating compounds to A-form and protonated form of poly(rC).poly(rG): spectroscopic and viscometric study.

Polymorphic RNA conformations may serve as potential targets for structure specific antiviral agents. As an initial step in the development of such drugs, the interaction of a wide variety of compounds which are characterized to bind to DNA through classical or partial intercalation or by mechanism of groove binding, with the A-form and the protonated form of poly(rC).poly(rG), been evaluated by multifaceted spectroscopic and viscometric techniques. Results of this study suggest that (i) ethidium intercalates to the A-form of RNA, but does not intercalate to the protonated form, (ii) methylene blue intercalates to the protonated form of the RNA but does not intercalate to the A-form, (iii) actinomycin D does not bind to either conformations of the RNA, and (iv) berberine binds to the protonated form by partial intercalation process, while its binding to the A-form is very weak. The DNA groove binder distamycin A has much higher affinity to the protonated form of the RNA compared to the A-form and binds to both structures by non-intercalative mechanism. We conclude that the binding affinity characteristics of these DNA binding molecules to the RNA conformations are vastly different and may serve as data for the development of RNA based antiviral drugs.

Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study.

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.