R. Malathi

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: A study of DNA-directed toxicity enlightened by time correlated single photon counting

Abstract Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylx-anthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A1 and higher lifetime component A are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A was increased by 7–8% and 17–37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1–2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity

Increased DNA-binding activity of cis-1,1-cyclobutanedicarboxylatodiammineplatinum(II) (carboplatin) in the presence of nucleophiles and human breast cancer MCF-7 cell cytoplasmic extracts: activation theory revisited

The molecular mechanism of carboplatin [cis-1,1-cyclobutanedicarboxylatodiammineplatinum(II)] activation is still unresolved. We studied the binding of carboplatin to calf thymus DNA in the presence of thiourea, glutathione, and human breast cancer MCF-7 cell cytoplasmic extracts by measurement of DNA-dependent ethidium bromide fluorescence and atomic absorption spectroscopy. After a 96-hr period of reaction, the decrease in the DNA-dependent fluorescence yield of ethidium bromide due to the formation of platinum (Pt)-DNA adducts increased significantly in the presence of thiourea (6-fold) and glutathione (3- to 4-fold) as compared to the controls in the absence of the nucleophiles. There was also a marked elevation in the levels of platinum incorporated into DNA, measured by atomic absorption spectroscopy (2- to 3-fold and 5- to 7-fold for thiourea and glutathione, respectively). More remarkably, the Pt-DNA adducts formed in the presence of cytoplasmic extracts of MCF-7 human breast cancer cells also showed similar results in a dose-related fashion. Carboplatin, therefore, displayed a characteristic increase in DNA binding/damaging in the presence of the very same S-containing nucleophiles that showed the expected quenching effects in the case of cisplatin [cis-diamminedichloroplatinum (II)]. We propose a nucleophile-facilitated release of the active species of carboplatin prior to binding with DNA.

Modulation of DNA intercalation by resveratrol and genistein

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10–100 μM and 10–150 μM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 μM and 100 μM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (τ2, A2) decreased with the subsequent increase in smaller component or constituent of free dye (τ1, A1) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.

RNA Binding Efficacy of Theophylline, Theobromine and Caffeine

Abstract The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 ± 5%), whereas moderate and comparatively less binding activity for theobromine (45 ± 5%) and caffeine (30 ± 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm−1), theobromine (3379.8 cm− 1) and caffeine (3343 cm−1) as compared to the free RNA (3341.6 cm−1). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (υC=O) of both drug (υC=O=1718, 1666 cm−1) as well as RNA (υC=O=1699, 1658 cm−1) disappeared and a new vibration band appeared around 1703 cm−1, indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theo- bromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

Introns and protein revolution- An analysis of the exon/intron organisation of actin genes

A catalogue of intron positions obtained from a large number of actin genes has been compiled with a view to understanding the possible origin of intervening sequences. Actins are ubiquitous proteins conserved in evolution and an analysis of their gene structures from various organisms has revealed that there may be at least 25 intron positions distributed at different positions in the coding regions. A comparison of intron positions from a wide range of organisms from that of yeast to human actins shows that introns could be more ancestral in origin. The conservation in the observed intron patterns within the different tissue types hints at a possible functional significance of introns in present day actin genes.

Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline, Theobromine and Caffeine) Binding with DNA

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg2+ and during helix-coil transitions of DNA by temperature (Tm) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophyllineu200a=u200a3.5×103 M−1, DNA-theobromineu200a=u200a1.1×103 M−1, and DNA-Caffeineu200a=u200a3.8×103 M−1. On the other hand Tm/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg2+, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg2+. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.

Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes -- a comparative study of group-I intron structures.

In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.

From RNA world to Protein: An eagle's eye view of the Role of Guanosine in Tracing the Antiquity of the Intron

A popular theory/idea is that first biocatalysts were made up of RNA and not proteins and that RNA first promoted sophisticated reactions required for life with the help of metals, pyridines, aminoacids and small molecule cofactors and thus must have been capable of both functions of the genetic material and an enzyme. DNA eventually replaced RNA as the genetic polymer and protein took over as the biocatalyst. One of the accepted proposition is that RNA world began with ribozymes that are capable of self-replicating, polymerase activity and also capable of other enzymatic activities (1,2) and to achieve the transition to the present nucleicacidprotein world, RNA must have been able to promote protein synthesis and other metabolic reactions. Ribozymes, some of which are capable of selfsplicing as in group I intron and nuclease activity of the RNA component of tRNA processing enzyme RNase P might be a natural choice for analysis as they might offer some clues to evolution of RNA-protein today from a prebiotic RNA world. In order to answer some of these questions regarding the RNA world hypothesis, and to trace the evolutionary pathway we have looked carefully into the detailed mechanism of RNA splicing that could have presumably started with primordial self-splicing RNA as found in group I & group II introns (in the absence of protein) and followed by enzyme mediated splicing as found in present day pre-mRNAs, prerRNAs and pre-tRNAs a situation that could truly represent a transition from a protein free RNA to contemporary protein-RNA interaction. Hence it would be of interest to explore, for instance the self-splicing group I intron ribozymes which are capable of replication, polymerisation etc., wherein the intron removal was catalyzed by itself in the absence of protein could be considered as the starting point which slowly got modified to the present day RNA splicing mechanism assisted by proteins and metals.

Analysis of Structure, Function, and Evolutionary Origin of the Ob Gene Product—Leptin

Abstract Leptin, the ob gene product, is a 167 amino acid polypeptide known to play a key role in regulating the fat stores of the body and is found in all eukaryotes, including mammals, aves, and also in invertebrates. To gain insight into the structure-function relation and origin of leptin, we have analyzed the amino acid sequence of leptin from 23 species by computing the frequency of occurrence of amino acids, their secondary structure, sequence homology, et cetera. Extensive conservation is observed within the leptin sequences of all the species, suggesting an evolutionary relatedness among them. It is interesting to note that human leptin shares a very high degree of homology with gorilla, chimpanzee, and orangutan indicative of a common function of leptin in them. Analysis of the codon bias in leptin from 11 species reveals that sminthopsis shows highest variation compared to human while less variation is observed in chimpanzee and orangutan, possibly reflecting the closeness in their evolution. Thus, understanding leptins three-dimensional structure along with primary and secondary structure might enable us to understand the functional role played by this multifaceted adipocyte derived protein.

Backbone Conformation in Nucleic Acids: An Analysis of Local Helicity through Heminucleotide Scheme and a Proposal for a Unified Conformational Plot

A relationship has been established to express the local helicity of a polynucleotide backbone directly in terms of the virtual bonds spanning the conformationally equivalent heminucleotide repeats, with a view to provide a better understanding of the cumulative effects of all the chemical bond rotational variations on local helicity. Using this, an analysis made with a few oligodeoxynucleotide crystal structures clearly brings forth that it is the concerted movements manifested in the near neighbour correlations between the pair of chemical bonds C4-C5 and P-O5 and C4-C3 and P-O3 of the 5 and 3 heminucleotides respectively that are primarily responsible for the observed non-uniform helical twists both in A and B type helical backbones. That these need not be restricted to oligodeoxynucleotides but may be a feature of oligoribonucleotides backbone also is shown from an analysis of helical segments of yeast tRNA(Phe). A proposal of a unified or a grand two dimensional conformational plot which would help visualise succinctly the overall effect of the variations in all the repeating six chemical bonds of a polynucleotide backbone is made. Apart from considerable simplification, the plot affords identification on it regions characteristic of helical, and loop and bend conformations of nucleic acid backbone chain.