In Silico Identification of Potential Quadruplex Forming Sequences in LncRNAs of Cervical Cancer.

Long non-coding RNAs (lncRNAs) have emerged as auxiliary regulators of gene expression influencing tumor microenvironment, metastasis and radio-resistance in cancer. The presence of lncRNA in extracellular fluids makes them promising diagnostic markers. LncRNAs deploy higher-order structures to facilitate a complex range of functions. Among such structures, G-quadruplexes (G4s) can be detected or targeted by small molecular probes to drive theranostic applications. The in vitro identification of G4 formation in lncRNAs can be a tedious and expensive proposition. Bioinformatics-driven strategies can provide comprehensive and economic alternatives in conjunction with suitable experimental validation. We propose a pipeline to identify G4-forming sequences, protein partners and biological functions associated with dysregulated lncRNAs in cervical cancer. We identified 17 lncRNA clusters which possess transcripts that can fold into a G4 structure. We confirmed in vitro G4 formation in the four biologically active isoforms of SNHG20, MEG3, CRNDE and LINP1 by Circular Dichroism spectroscopy and Thioflavin-T-assisted fluorescence spectroscopy and reverse-transcriptase stop assay. Gene expression data demonstrated that these four lncRNAs can be potential prognostic biomarkers of cervical cancer. Two approaches were employed for identifying G4 specific protein partners for these lncRNAs and FMR2 was a potential interacting partner for all four clusters. We report a detailed investigation of G4 formation in lncRNAs that are dysregulated in cervical cancer. LncRNAs MEG3, CRNDE, LINP1 and SNHG20 are shown to influence cervical cancer progression and we report G4 specific protein partners for these lncRNAs. The protein partners and G4s predicted in lncRNAs can be exploited for theranostic objectives.

Chimeric nanoparticles for targeting mitochondria in cancer cells.

Mitochondrial dysfunction is implicated in myriad diseases, including cancer. Subsequently, targeting mitochondrial DNA (mt-DNA) in cancer cells has emerged as an unorthodox strategy for anti-cancer therapy. However, approaches targeting only one component of the mitochondrial "central dogma" can be evaded by cancer cells through various mechanisms. To address this, herein, we have engineered mitochondria-targeting cholesterol-based chimeric nanoparticles (mt-CNPs) consisting of cisplatin, camptothecin, and tigecycline, which can simultaneously impair mt-DNA, mitochondrial topoisomerase I (mt-Top1), and mitochondrial ribosomes. mt-CNPs were characterized as being positively charged, spherical in shape, and 187 nm in diameter. Confocal microscopy confirmed that mt-CNPs efficiently localized into the mitochondria of A549 lung cancer cells within 6 h, followed by mitochondrial morphology damage and the subsequent generation of reactive oxygen species (ROS). mt-CNPs showed remarkable cancer-cell killing abilities compared to free-drug combinations in A549 (lung), HeLa (cervical), and MCF7 (breast) cancer cells. These mitochondria-targeting lipidic chimeric nanoparticles could be explored further to impair multiple targets in mitochondria, helping researchers to gain an understanding of mitochondrial translational and transcriptional machinery and to develop new strategies for cancer therapy.

Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay.

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.

Nanoparticle-Mediated Routing of Antibiotics into Mitochondria in Cancer Cells.

In recent years, antibiotics have emerged as alternative medicines in cancer therapy due to their capability of mitochondrial dysfunction in cancer cells. However, antibiotics render collateral damage in noncancerous cells by targeting mitochondrial transcription and translational machinery. To address this, herein, we have engineered three different mitochondria-targeted cationic antibiotic (tigecycline)-loaded nanoparticles from cholesterol conjugates. Dynamic light scattering and electron microscopy confirmed the spherical morphology and a less than 200 nm hydrodynamic diameter for these nanoparticles. The triphenylphosphine-coated tigecycline-loaded nanoparticle (Mito-TPP-Tig-NP) was shown to be homed into the mitochondria of A549 lung cancer cells compared to the other cationic nanoparticles. These Mito-TPP-Tig-NPs indeed triggered mitochondrial morphology damage and generation of reactive oxygen species (ROS). All the mitochondria-targeted tigecycline-loaded nanoparticles showed improved cancer cell killing ability in A549 and HeLa cervical cancer cells compared to free tigecycline. Moreover, Mito-TPP-Tig-NPs showed much less toxicity toward noncancerous human embryonic kidney cells (HEK293) compared to free tigecycline. These antibiotic-loaded mitochondria-targeted nanoparticles can open up an avenue toward anticancer therapy.

Small Molecule Fluorescent Probes for G- Quadruplex Visualization as Potential Cancer Theranostic Agents.

G-quadruplexes have gained prominence over the past two decades for their role in gene regulation, control of anti-tumour activity and ageing. The physiological relevance and significance of these non-canonical structures in the context of cancer has been reviewed several times. Putative roles of G-quadruplexes in cancer prognosis and pathogenesis have spurred the search for small molecule ligands that are capable of binding and modulating the effect of such structures. On a related theme, small molecule fluorescent probes have emerged that are capable of selective recognition of G-quadruplex structures. These have opened up the possibility of direct visualization and tracking of such structures. In this review we outline recent developments on G-quadruplex specific small molecule fluorescent probes for visualizing G-quadruplexes. The molecules represent a variety of structural scaffolds, mechanism of quadruplex-recognition and fluorescence signal transduction. Quadruplex selectivity and in vivo imaging potential of these molecules places them uniquely as quadruplex-theranostic agents in the predominantly cancer therapeutic context of quadruplex-selective ligands.

Interaction of a dimeric carbocyanine dye aggregate with bovine serum albumin in non-aggregated and aggregated forms.

The interaction of fluorescent dyes with serum proteins has garnered significant interest owing to potential for non-covalent labeling and imaging applications. In this work, dimeric benzothiazole-based trimethine cyanine dyes are synthesized and their interaction with bovine serum albumin studied. The dimeric cyanine dyes mainly exist as H-dimers and H-aggregates in aqueous solution. A combination of absorbance, fluorescence, circular dichroism spectroscopy and atomic force and fluorescence microscopy indicate the formation of dye-BSA complexes. Binding of one of the dimeric dyes on BSA with a Ka of 1.49×105M-1 results in disruption of dye self-aggregates and unfolding of the dyes into the monomeric or open conformation. Fluorescence enhancement experienced by the dimeric dyes upon interaction with BSA is superior to that registered by Thioflavin T. Surfactant SDS has been used to further tune the self-aggregation of the dimeric dye resulting in a 200-fold fluorescence enhancement in presence of BSA. Interaction of a dimeric dye with BSA under conditions favoring protein aggregation is found to result in faster dye binding and the resulting fluorescence enhancement is easily visualized by fluorescence microscopy. The interaction of a dimeric cyanine dye aggregate with BSA is promising for non-covalent labeling applications in sharp contrast to the monomeric dye counterpart.