Ambadas B. Rode

Tuning Riboswitch‐Mediated Gene Regulation by Rational Control of Aptamer Ligand Binding Properties

Riboswitch-mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN-specific riboswitches were performed. The strong dependence of the dissociation rate (52.6-fold) and affinity (100-fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base-pair composition and context, is the tuning region of the FMN-specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31-fold difference in the half-maximal effective concentration (EC50) corresponded to a 11.6-fold difference in the dissociation constant (K(D)) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.

5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells

In the present study, we investigated the signaling pathways implicated in the induction of apoptosis by two modified nucleosides, 5-phenylselenyl-methyl-2′-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2′-deoxyuridine (MeSe-T), using human cancer cell lines. The induction of apoptosis was associated with proteolytic activation of caspase-3 and -9, PARP cleavage, and decreased levels of IAP family members, including c-IAP-1 and c-IAP-2, but had no effect on XIAP and survivin. PhSe-T and MeSe-T also enhanced the activities of caspase-2 and -8, Bid cleavage, and the conformational activation of Bax. Additionally, nucleoside derivative-induced apoptosis was inhibited by the selective inhibitors of caspase-2, -3, -8, and -9 and also by si-RNAs against caspase-2, -3, -8, and -9; however, inhibition of caspase-2 and -3 was more effective at preventing apoptosis than inhibition of caspase-8 and -9. Moreover, the inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk or by the knockdown of protein expression using siRNA suppressed nucleoside derivative-induced caspase-3 activation, but not vice versa. PhSe-T and MeSe-T also induced a Δψm loss via a CsA-insensitive mechanism, ROS production, and DNA damage, including strand breaks. Moreover, ROS scavengers such as NAC, tiron, and quercetin inhibited nucleoside derivative-induced ROS generation and apoptosis by blocking the sequential activation of caspase-2 and -3, indicating the role of ROS in caspase-2-mediated apoptosis. Taken together, these results indicate that caspase-2 acts upstream of caspase-3 and that caspase-2 functions in response to DNA damage in both PhSe-T- and MeSe-T-induced apoptosis. Our results also suggest that ROS are critical regulators of the sequential activation of caspase-2 and -3 in nucleoside derivative-treated cancer cells.

Real-Time Monitoring of G-Quadruplex Formation during Transcription

Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of G-quadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation.

Potent radiosensitizing agents: 5-methylselenyl- and 5-phenylselenyl-methyl-2'-deoxyuridine.

This Letter describes the novel radiosensitizing agents based on nucleoside base modification. In addition to the known 5-phenylselenide derivative, 5-methylselenide modified thymidine, which has a van der Waals radius smaller than the phenyl group, was newly synthesized. The similar monomer activity of 5-methylselenide derivative under oxidation condition was confirmed by NMR experiments. The cytotoxicity tests and radiosensitizing experiments of both compounds were carried out using the H460 lung cancer cell line. Both the 5-phenylselenide and the 5-methylselenide derivatives showed a relatively low toxicity to the cells. However, in combination with γ-radiolysis, both exerted good radiosensitizing effects to the lung cancer cell lines in vitro. This result confirms that 5-methylselenide modified thymidine could be a useful candidate as a potential radiosensitizing agent in vivo.

H2O2/Phosphonium Ionic Liquid: An Efficient and Simple Approach for Benzyl Halides Oxidation

Abstract: An efficient oxidation of benzyl halide has been done using aqueous hydrogen peroxide (30%) in trihexyl(tetradecyl)phosphonium-tetrafluroborate ionic liquid the protocol is simple mild offering excellent yield of product. Keywords: Benzyl halides, carbonyl compounds, aq. hydrogen peroxide (30%), ionic liquids. INTRODUCTION Benzyl halides oxidation is an important tool in synthetic organic chemistry. Different reagents have been used for this transformation [1, 2]. In last two decades applications of hypervalent iodine have been proliferating in synthetic organic chemistry [3], among various hypervalent iodine reagents o- iodoxy benzoic acid (IBX) is used as versatile oxidizing agent [4], because of its mild, selective, efficient and eco-friendly properties and operational simplicity. Oxygen mediated oxidation of alkenes using Nafion supported Platinum (II) Terpyridyl Acetylide complex is also used in Photosensitization reaction [5]. Non-volatile ionic liquids have been recognized as environmentally benign alternatives to volatile organic solvents [6].

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.