Biophysical Characterization of the Strong Stabilization of the RNA Triplex poly(U)•poly(A)*poly(U) by 9-O-(ω-amino) Alkyl Ether Berberine Analogs

Background: Binding of two 9-O-(v-amino) alkyl ether berberine analogs BC1 and BC2 to the RNA triplex poly(U)Npoly(A)*poly(U) was studied by various biophysical techniques. Methodology/Principal Findings: Berberine analogs bind to the RNA triplex non-cooperatively. The affinity of binding was remarkably high by about 5 and 15 times, respectively, for BC1 and BC2 compared to berberine. The site size for the binding was around 4.3 for all. Based on ferrocyanide quenching, fluorescence polarization, quantum yield values and viscosity results a strong intercalative binding of BC1 and BC2 to the RNA triplex has been demonstrated. BC1 and BC2 stabilized the Hoogsteen base paired third strand by about 18.1 and 20.5uC compared to a 17.5uC stabilization by berberine. The binding was entropy driven compared to the enthalpy driven binding of berbeine, most likely due to additional contacts within the grooves of the triplex and disruption of the water structure by the alkyl side chain. Conclusions/Significance: Remarkably higher binding affinity and stabilization effect of the RNA triplex by the amino alkyl berberine analogs was achieved compared to berberine. The length of the alkyl side chain influence in the triplex stabilization phenomena

Biophysical studies on the interaction of isoquinoline alkaloids and analogues with nucleic acids

Deoxyribonucleic acid, generally referred to as DNA by its acronym, is the basis of life, which contains the information for the development and functioning of all the living organisms. The DNA research began in 1868, when Swiss physiological chemist Friedrich Miescher first identified what he called “nuclein” inside the nuclei of human white blood cells. In 1889, R. Altmann separated nuclein from protein and because of its acidic character he named it nucleic acid. Phoebus Levene identified the components of DNA and showed that they were linked in the order phosphate- sugar-base to form units which he referred as nucleotide and suggested that the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups, which is the ‘backbone’ of the molecule. The role of DNA in heredity was recognized in 1944, when Avery and co-workers published their famous result that DNA and not proteins were the carriers of genetic information (Avery et al., 1944). The complementary base-pair rule was found by Chargaff in 1950 (Chargaff et al., 1950). A milestone in DNA research was the double helix structure which was proposed by Watson and Crick in 1953 based upon the X-ray fiber diffraction data from fibres of DNA obtained by Rosalind Franklin (Watson and Crick, 1953). From the double helical structure, it was immediately obvious how information could pass from one generation to the next by synthesis of DNA complementary strands from parent strands. In 1962 they received the Nobel Prize in Chemistry for the discovery along with the crystallographer Maurice Wilkins. The first published account of the directed chemical synthesis of an oligonucleotide occurred in 1955 when Michelson and Todd reported the preparation of a dithymidinyl nucleotide (Michelson and Todd, 1955). In the late 1950’s Khorana and his group were able to synthesize oligoribonucleotides that were used to confirm the Genetic Code. In 1968, Khorana received the Nobel Prize in Physiology or Medicine for their interpretation of the genetic code and its function in protein synthesis. Khorana’s method was revolutionary at the time and produced a truly remarkable feat: the synthesis of an active 72-mer tRNA molecule, which was published in Nature (Agarwal et al., 1970). However, it was not until the late 1970’s that the development of DNA research became explosive, when synthetic DNA fragments became commercially available. Pohl and Jovin first observed the salt-induced cooperative conformational changes of the synthetic DNA poly(dG-dC).poly(dG-dC) duplex, from a right-handed helix to left handed helix from the circular dichroism study (Pohl and Jovin, 1972)

Recent Advances in Nucleic Acid Binding Aspects of Berberine Analogs and Implications for Drug Design

Berberine is one of the most widely known alkaloids belonging to the protoberberine group exhibiting myriad therapeutic properties. The anticancer potency of berberine appears to derive from its multiple actions including strong interaction with nucleic acids exhibiting adenine-thymine base pair specificity, inhibition of the enzymes topoisomerases and telomerases, and stabilizing the quadruplex structures. It was realized that the development of berberine as a potential anticancer agent necessitates enhancing its nucleic acid binding efficacy through appropriate structural modifications.More recently a number of such approaches have been attempted in various laboratories with great success. Several derivatives have been synthesized mostly with substitutions at the 8, 9 and 13 positions of the isoquinoline chromophore, and studied for enhanced nucleic acid binding activity. In this article, we present an up to date review of the details of the interaction of berberine and several of its important synthetic 8, 9 and 13 substituted derivatives with various nucleic acid structures reported recently. These studies provide interesting knowledge on the mode, mechanism, sequence and structural specificity of the binding of berberine derivatives and correlate structural and energetic aspects of the interaction providing better understanding of the structure- activity relations for designing and development of berberine based therapeutic agents with higher efficacy and therapeutic potential

Influence of Flexible ``omega'' on the Activity of E-coli RNA Polymerase: A Thermodynamic Analysis

The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, omega. Despite the evolutionary conservation of omega and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for omega) are viable due to the association of RNAP with the global chaperone protein GroEL. With an aim to get better insight into the structure and functional role of , we isolated a dominant negative mutant of omega (omega(6)), which is predominantly a-helical, in contrast to largely unstructured native omega, and then studied its assembly with reconstituted core1 (alpha(2)beta beta') by a biophysical approach. The mutant showed higher binding affinity compared to native omega. We observed that the interaction between core1 and omega(6) is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in omega(6) makes it more rigid, the plasticity of the interacting domain formed by omega(6) and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (omega(6)) affects initiation of transcription. However, once preinitiated, the complex elongates the RNA chain efficiently. The initiation of transcription requires recognition of appropriate-factors by the core enzyme (core2: alpha(2)beta beta'omega). We found that the altered core enzyme (alpha(2)beta beta'omega(6)) with mutant omega showed a decrease in binding affinity to the sigma-factors (sigma(70), sigma(32) and sigma(38)) compared to that of the core enzyme containing native omega. In the absence of unstructured omega, the association of sigma-factors to the core is less efficient, suggesting that the flexible native omega plays a direct role in sigma-factor recruitment

The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: Photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction

The interaction of the natural benzophenanthridine alkaloid chelerythrine with DNA was studied by spectroscopy, viscometry and calorimetry techniques. The absorbance and fluorescence properties of the alkaloid were remarkably modified upon binding to DNA and the interaction was found to be cooperative. The mode of binding was principally by intercalation as revealed from viscosity studies and supported from fluorescence quenching, and polarization results. The binding remarkably stabilized the DNA structure against thermal strand separation. The binding induced conformational changes in the B-form structure of the DNA and the bound alkaloid molecule acquired induced circular dichroism. The binding affinity values obtained from spectroscopy, fluorescence polarization (and anisotropy) and calorimetry were in agreement with each other. The binding was exothermic, characterized by negative enthalpy and positive entropy change and exhibited enthalpy–entropy compensation phenomenon. The heat capacity changes of the binding revealed hydrophobic contribution to the binding. Molecular aspects of the interaction characterized by the involvement of multiple weak noncovalent forces are presented

Stepwise Unfolding of Bovine and Human Serum Albumin by an Anionic Surfactant: An Investigation Using the Proton Transfer Probe Norharmane

Interactions of the anionic surfactant sodium dodecyl sulfate (SDS) with thetransport proteins bovine serum albumin (BSA) and human serum albumin (HSA) have been divulged using an external photoinduced proton transfer probe, norharmane (NHM).Steady-state fluorometry, time-resolved measurements, micropolarity analysis, circular dichroism (CD), and isothermal titration calorimetry (ITC) have been exploited for the study. With the gradual addition of SDS to the probe-bound proteins, the fluorometric responses of the different prototropic species of NHM exhibit an opposite pattern as to that observed while NHM binds to the proteins. The study reveals a sequential unfolding of the serum proteins with the gradual addition of SDS. ITC measures the heat changes associated with each step of the unfolding. ITC experiments, carried out at two different pH’s, elucidate the nature of interaction between SDS and the two serum proteins. At a very high concentration of SDS, the external probe (NHM) is found to be dislodged from the protein environments to bind to the SDS micellar medium

Variation of the relative fluorescence quantum yields, anisotropy and visocisty.

<p>(A) Variation of the relative fluorescence quantum yield of BC (▪), BC1 (•), and BC2 (▴) in the presence of poly(U)_•<b>.</b>poly(A)<b>_*</b>poly(U) as a function of excitation wavelength. (B) A plot of the variation of anisotropy values versus P/D ratio for the complexation of BC (▪), BC1 (•), BC2 (▴) with poly(U)_•poly(A)<b>_*</b>poly(U). (C) A plot of increase in helix contour length (L/Lo) versus r for the complexation of BC (▪), BC1 (•), BC2 (▴) with poly(U)_•poly(A)<b>_*</b>poly(U) at 25±0.5°C.</p

Absorption spectral change of alkaloids in presence of triplex.

<p>(A) BC (B) BC1 (C) BC2 each of 5.0 μM treated with increasing concentrations of poly(U)_•poly(A)<b>_*</b>poly(U). P/D saturation for BC, BC1 and BC2 were 28.0, 20.0 and 15.0, respectively. Inset: respective non-cooperative Scatchard isotherms of the binding.</p

Fluorescence spectra of BC and BC analogs in presence of triplex.

<p>(A) BC (B) BC1 (C) BC2 (2.0 μM each) treated with increasing concentration of RNA triplex. P/D saturation for BC, BC1 and BC2 were 32.0, 24.0 and 18.0, respectively. Inset: respective non-cooperative Scatchard plots of binding.</p

ITC Derived Thermodynamic parameters for the binding of BC, BC1 and BC2 with poly(U)_•poly(A)_*poly(U) triplex at 25°C.^a

a<p>All the data in this table are derived from ITC experiments and are average of four determinations. <i>K_a</i> and Δ<i>H</i>^o values were determined from ITC profiles fitting to Origin 7 software as described in the text. n is the site size which is the reciprocal of the stoichiometry N. The values of Δ<i>G</i>^o and TΔ<i>S</i>^o were determined using the equations Δ<i>G</i>^o  =  − RTln <i>K_a</i> and Δ<i>G</i>^o<i> = </i>Δ<i>H</i>^o − TΔ<i>S</i>^o. All the ITC profiles were fit to a model of single binding site.</p