Mahima Kaushik

Structural Diversity and Specific Recognition of Four Stranded G-Quadruplex DNA

Structural multitude of nucleic acids serves basis for its multiple merits and applications. During structural transitions, significant to perform respective cellular functions, these DNA forms can vary from the single stranded to multi-stranded species. Hence, beyond the image of a monotonous DNA double-helix, there is now increasing interest in other polymorphic/ multi-stranded forms, the roles they may play in vivo and their potential use in therapeutics. Distinct guanine-rich nucleic acid sequences readily form a structurally diverse four-stranded architecture called G-quadruplexes. In addition to their presence at physical ends of chromosomes called telomeres, occurrence of these structural motifs in the upstream promoter regions of a number of genes, oncogenes and near transcription start sites, highlights that G-quadruplexes are involved in regulation of gene expression. Cancer cells typically possess shorter telomeres and have telomerase activity greatly exceeding that of normal cells. These differences create an opportunity to use anticancer therapies targeting telomerase and telomeres. The ability of small molecules to interact with and presumably stabilize G-quadruplex structures as a means of inhibiting telomerase has been a major drug design effort. Ligands, capable of interacting with four-stranded G-quadruplex have been generated. The discovery of proteins including transcription factors, recognizing G-quadruplexes, and conferring stabilization or unfolding them in biological systems, again makes G-quadruplexes, biologically pertinent structures. This review is an attempt to summarize the rapidly evolving literature exploring the amazing polymorphism of G-quadruplexes, and understanding their structure-specific-recognition and biological relevance, keeping in mind that G-tetraplexes are not only important drug targets, but may also act as gene regulatory elements. A pertinent detail of the challenges towards the rational design of structure-specific novel drugs has also been discussed.

A bouquet of DNA structures: Emerging diversity

Structural polymorphism of DNA has constantly been evolving from the time of illustration of the double helical model of DNA by Watson and Crick. A variety of non-canonical DNA structures have constantly been documented across the globe. DNA attracted worldwide attention as a carrier of genetic information. In addition to the classical Watson–Crick duplex, DNA can actually adopt diverse structures during its active participation in cellular processes like replication, transcription, recombination and repair. Structures like hairpin, cruciform, triplex, G-triplex, quadruplex, i-motif and other alternative non-canonical DNA structures have been studied at length and have also shown their in vivo occurrence. This review mainly focuses on non-canonical structures adopted by DNA oligonucleotides which have certain prerequisites for their formation in terms of sequence, its length, number and orientation of strands along with varied solution conditions. This conformational polymorphism of DNA might be the basis of different functional properties of a specific set of DNA sequences, further giving some insights for various extremely complicated biological phenomena. Many of these structures have already shown their linkages with diseases like cancer and genetic disorders, hence making them an extremely striking target for structure-specific drug designing and therapeutic applications.

Structural polymorphism at LCR and its role in beta-globin gene regulation

Information on the secondary structures and conformational manifestations of eukaryotic DNA and their biological significance with reference to gene regulation and expression is limited. The human beta-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I-hypersensitive sites (HSs). Since these HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. Mutations and SNPs may stabilize or destabilize the local secondary structures, affecting the gene expression by changes in the protein-DNA recognition patterns. Various palindromic or quasi-palindromic segments within LCR, could cause structural polymorphism and geometrical switching of DNA. This emphasizes the importance of understanding of the sequence-dependent variations of the DNA structure. Such structural motifs might act as regulatory elements. The local conformational variability of a DNA segment or action of a DNA specific protein is key to create and maintain active chromatin domains and affect transcription of various tissue specific beta-globin genes. We, summarize here the current status of beta-globin LCR structure and function. Further structural studies at molecular level and functional genomics might solve the regulatory puzzles that control the beta-globin gene locus.

Temperature induced hyperchromism exhibited by Hoechst 33258: evidence of drug aggregation from UV-melting method

UV-thermal denaturation is a simple optical method widely employed for determination of DNA stability and interaction with ligands. Thermal denaturation of DNA and DNA-ligand complex is usually monitored at 260 nm. These data are generally presented as a function of the absorption increase of DNA alone with no consideration of the temperature dependent hyperchromism of the free ligand. Since not every ligand has absorption at 260 nm, usually this property of the ligand is ignored. Here, we report the temperature dependent hyperchromicity exhibited by Hoechst 33258 in the presence and absence of DNA. The presence of Hoechst, added to the duplex (monophasic profile, T(m)=75 degrees C) in various ratios generates a new transition at lower temperature displaying biphasic thermal transition profiles. We attributed this new transition (hyperchromic), a mere contribution from Hoechst, which might exist in aggregated forms. The extent of drug aggregation/self-association is concentration dependent. We suggest that prior to UV-melting studies the thermal dependence of the free ligand should be investigated.

Protein engineering and de novo designing of a biocatalyst.

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging.

Structure-Specific Ligand Recognition of Multistranded DNA Structures.

Structural polymorphism is an extremely significant phenomenon of nucleic acids, in which DNA and RNA oligonucleotide sequences are able to adapt various canonical, alternative and multistranded structures. These alternative forms of DNA and RNA have an enormous potential of participating in various cellular processes by recognizing ligands such as proteins, drugs and metal ions in a sequence and structure-specific manner. Such DNA-ligand interactions prove to be highly beneficial when exploited for therapeutic purposes. Many of these DNA/ RNA structures recognizing drugs have already proved their potential as anticancer, antibacterial, anthelmintic and antiviral properties. Over the last 2-3 decades, many mechanisms of DNA-drug interactions have been documented, but still many other new mechanisms are being explored. Designing new drugs with improved efficacy and specificity is of prime concern for all researchers which not only deals with the experiments related to synthesizing drugs, but also takes care of searching novel routes or agents for administration or delivery of these therapeutic agents by increasing their nuclear and cellular uptake. This review aims at explaining the structural polymorphs/ multistranded DNA structures and their interactions with pharmaceutical drugs in a structure-specific manner, along with their modes of interactions and biological relevance. This detailed overview of multistranded DNA structures and interacting drugs might further facilitate our understanding about molecular targets and drug development in a more precise manner for the larger benefit of mankind.

Multiple dimensions of functional relevance of genosensors

ABSTRACT Biosensors having the recognition element as DNA/RNA oligon-ucleotides are known as genosensors, which have been widely utilized for many applications including clinical diagnostic tool for diseases like cancer and a range of infectious diseases. This review aims to discuss the vast repertoire of nanomaterial based genosensors, their designing and numerous applications, including their usage in food quality assessment, environmental monitoring, as aptasensors for the recognition of nucleosides, as biomarkers for the identification of DNA methylation in epigenetics and molecular beacon nano-sensors for probing the living cancer cells etc. Genosensors are of utmost significance for solving various puzzles related to cellular processes and their control mechanisms.

Spectroscopic Studies of the Binding Interactions of PhenothiazininumDyes (Thionine Acetate, Azure A and Azure B) with Calf-thymus DNA

The double helical structure of DNA offers various binding sites for the interaction of ligands or proteins. Interactions using minor groove, major groove, and through intercalation are the major types of binding mechanisms of DNA-ligand interactions. The lowering in the absorption intensity along with bathochromic shift is the indication of intercalation binding mode of the dye into the base pairs of the DNA. In this study, the interaction of phenothiazine dyes with calf-thymus DNA (ctDNA) in physiological buffer (pH 7.4) was studied using UV-visible, fluorescence, circular dichroism (CD), and UV-thermal denaturation spectroscopy. The binding constants were calculated at different temperatures with the help of fluorescence spectroscopy. CD signals signify that B-form of DNA might become more compact, upon binding of the dyes. Also, induced circular dichroism is observed which confirms the dye-DNA complex formation. Stabilization of DNA double helix upon binding with dyes was confirmed by the increase in Tm of ctDNA. Based on thermal melting profiles, it was found that thionine acetate is most promising in stabilizing the DNA double helix, in comparison to other two dyes. Also, binding constants calculated by fluorescence is in accordance with the thermal melting analysis. These results are indicative of the intercalation binding mode between dyes and the DNA. The binding affinity of the dyes to DNA is found to be in order as thionine acetate > azure A > azure B. Such preliminary studies facilitate our understanding about various types of DNAligand interactions and provide clues for designing new and more effective drugs.

Differential structural status of the RNA counterpart of an undecamer quasi-palindromic DNA sequence present in LCR of human β-globin gene cluster

Our previous work on structural polymorphism shown at a single nucleotide polymorphism (SNP) (A→G) site located on HS4 region of locus control region (LCR) of β-globin gene has established a hairpin→duplex equilibrium corresponding to A→B like DNA transition (Kaushik M, Kukreti, R., Grover, D., Brahmachari, S.K. and Kukreti S. Nucleic Acids Res. 2003; Kaushik M, Kukreti S. Nucleic Acids Res. 2006). The G-allele of A→G SNP has been shown to be significantly associated with the occurrence of β-thalassemia. Considering the significance of this 11-nt long quasi-palindromic sequence [5′-TGGGG(G/A)CCCCA; HP(G/A)11] of β-globin gene LCR, we further explored the differential behavior of the same DNA sequence with its RNA counterpart, using various biophysical and biochemical techniques. In contrast to its DNA counterpart exhibiting a A→B structural transition and an equilibrium between duplex and hairpin forms, the studied RNA oligonucleotide sequence [5′-UGGGG(G/A)CCCCA; RHP(G/A)11] existed only in duplex form (A-conformation) and did not form hairpin. The single residue difference from A to G led to the unusual thermal stability of the RNA structure formed by the studied sequence. Since, naturally occurring mutations and various SNP sites may stabilize or destabilize the local DNA/RNA secondary structures, these structural transitions may affect the gene expression by a change in the protein–DNA recognition patterns.

Exploring the DNA damaging potential of chitosan and citrate-reduced gold nanoparticles: Physicochemical approach

Nanomaterials offer a wide range of biomedical applications including gene/drug delivery, biosensing and bioimaging. The cytotoxic and genotoxic potential of nanoparticles need to be thoroughly investigated before their biomedical usage. This study aims to investigate and compare the nanotoxicology of chitosan (CH-Au-Np) and citrate (CI-Au-Np) reduced gold nanoparticles via exploring their interaction with Calf thymus DNA (Ct-DNA) utilizing various physicochemical techniques. Structural characterization of these Nps was done using UV-Visible Spectroscopy and Transmission Electron Microscopy (TEM). Analysis of UV-Visible absorbance spectra indicates that interaction of CH-Au-Np with Ct-DNA causes destabilization of DNA by inducing significant structural and conformational changes in Ct-DNA in a concentration dependent manner, whereas there was negligible interaction between CI-Au-Np and Ct-DNA. These observations were further supported by the results of agarose gel mobility, UV-thermal melting, Circular Dichroism (CD), Dynamic Light Scattering (DLS) and TEM studies. Fluorescence spectral studies using acridine orange (AO) as a fluorescence probe and analysis of thermodynamic parameters reveal that the interactions between Ct-DNA and CH-Au-Np were mainly governed by Van der Waal interactions and Hydrogen bonding. An insightful understanding of genotoxicity induced by CH-Au-Np can be advantageous, as it may provide valuable anticancer approach for cytotoxic drug designing.