pH-dependent complex formation with TAR RNA and DNA: application towards logic gates.

Nucleic acid-based logic gates have shown great potential in biotechnology, medicine as well as diagnostics. Herein, we have constructed pH-responsive logic devices by utilizing HIV-1 TAR hairpins in combination with a thiazole peptide that exhibits turn-on fluorescence upon interacting with TAR RNA or DNA. Based on this, INHIBIT-AND and YES-INHIBIT-AND logic gates were constructed in parallel. The pH alteration leads to conformational changes of the hairpin structure, enabling the construction of a multi-reset reusable logic system which could be developed for in vitro sensing of the HIV-1 viral RNA.

Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+ and Mn2+ ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

Role of abiotic factors in enhancing the capacity of mangroves in reducing ocean acidification.

The present study investigated the effects of rising carbon dioxide levels in nature and the carbon sequestration potential of dominant mangrove species for reducing the toxic effects of ocean acidification. The study was conducted on the east coast of Odisha, in the western Bay of Bengal. To determine the effect of these ambient parameters on the absorption of carbon dioxide by the mangroves, water temperature, salinity, pH levels of seawater along with soil texture and pH, salinity expressed in electrical conductivity, compactness expressed in bulk density, and soil organic carbon were simultaneously monitored. The aboveground biomass and carbon of the selected species were studied for 2 consecutive years at 10 designated stations. The total carbon calculated for the study area varied from 242.50 ± 49.00 to 1321.29 ± 445.52 tons with a mean of 626.68 ± 174.81 tons for Bhitarkanika and Mahanadi mangrove chunks. This is equivalent to 2299.92 ± 641.55 tons of CO2 absorbed from the atmosphere. A total of 27 equations were selected as the best fit models for the study area. The equations between mangrove biomass and carbon along with aquatic and edaphic factors governing the pH of water and soil strongly support the positive influence of mangrove photosynthetic activity in shifting the equilibrium toward alkalinity. This calls for conservation of mangrove ecosystem to minimize the pace of acidification of estuarine water. The results indicate that Excoecariaagallocha and Avicennia marina as are the most capable species for combatting maximum carbon dioxide toxicity from the atmosphere; which will be helpful in REDD + programs and carbon-based payments for ecosystem services (PES).

G-Quadruplex-Binding Small Molecule Induces Synthetic Lethality in Breast Cancer Cells by Inhibiting c-MYC and BCL2 Expression.

Herein, a prolinamide-derived peptidomimetic that preferentially binds to c-MYC and BCL2 G-quadruplexes present in the promoter regions of apoptosis-related genes (c-MYC and BCL2) over other DNA quadruplexes are described. Biological assays, such as real-time quantitative reverse transcription, western blot, dual luciferase, and small interfering RNA knockdown assays, indicate that the ligand triggers a synthetic lethal interaction by simultaneously inhibiting the expression of c-MYC and BCL2 genes through their promoter G-quadruplexes. The ligand shows antiproliferative activity in MCF-7 cells that overexpress both MYC and BCL2 genes, in comparison to cells that overexpress either of the two. Moreover, the ligand induces S-phase cell-cycle arrest, DNA damage, and apoptosis in MCF-7 cells.

Drug-Drug Binary Solids of Nitrofurantoin and Trimethoprim: Crystal Engineering and Pharmaceutical Properties.

With the aim of developing multidrug solids through a tuned crystal engineering approach, we have selected two antiurinary infective drugs, namely, nitrofurantoin (NF) and trimethoprim (TMP) and isolated eight binary drug-drug solid solvates along with a nonsolvated cocrystal. Crystal structure analyses were performed for eight of these solids and rationalized in terms of known supramolecular synthons formed by pyrimidine, imide, and amine functionalities. Notably, the TMP-NF anhydrous cocrystal and its ionic cocrystal hydrate exhibit enhanced equilibrium solubilities compared to pure NF or the simple NF hydrate. Furthermore, the ionic cocrystal hydrate exhibits greater antibacterial activity against the Gram-negative bacteria, E. coli, compared to the parent TMP and NF at the lowest concentration of 3.9 µg/mL. This study indicates initial pathways using the cocrystal methodology that would help to eventually arrive at an antiurinary cocrystal with optimal properties.

Cell penetrating thiazole peptides inhibit c-MYC expression via site-specific targeting of c-MYC G-quadruplex.

The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5'- and 3'-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

Target-Directed Azide-Alkyne Cycloaddition for Assembling HIV-1 TAR RNA Binding Ligands.

The highly conserved HIV-1 transactivation response element (TAR) binds to the trans-activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat-TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV-1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin-tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole-linked thiazole peptidomimetic products have been isolated from the biotin-tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat-TAR interactions.

EC-tagging allows cell type-specific RNA analysis.

Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.

Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase.

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.

Mechanistic Studies on RNA Strand Scission from a C2'-Radical.

The C2'-carbon-hydrogen bond in ribonucleotides is significantly weaker than other carbohydrate carbon-hydrogen bonds in RNA or DNA. Independent generation of the C2'-uridine radical (1) in RNA oligonucleotides via Norrish type I photocleavage of a ketone-substituted nucleotide yields direct strand breaks via cleavage of the ß-phosphate. The reactivity of 1 in different sequences and under a variety of conditions suggests that the rate constant for strand scission is significantly greater than 106 s-1 at pH 7.2. The initially formed C2'-radical (1) is not trapped under a variety of conditions, consistent with computational studies ( Chem.-Eur. J. 2009 , 15 , 2394 ) that suggest that the barrier to strand scission is very low and that synchronous proton transfer from the 2'-hydroxyl to the departing phosphate group facilitates cleavage. The C2'-radical could be a significant contributor to RNA strand scission by the hydroxyl radical, particularly under anaerobic conditions where 1 can be produced from nucleobase radicals.

Structural features facilitating tumor cell targeting and internalization by bleomycin and its disaccharide.

We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide-cytotoxin conjugates.

Protein Synthesis with Ribosomes Selected for the Incorporation of ß-Amino Acids.

In an earlier study, ß³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different ß-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate ß-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the ß³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-ß-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-ß-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated ß-tyrosine moiety of ß³-puromycin. Also conducted were a selection of clones that are responsive to ß²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

Rapid RNA strand scission following C2'-hydrogen atom abstraction.

C2'-Nucleotide radicals have been proposed as key intermediates in direct strand break formation in RNA exposed to ionizing radiation. Uridin-2'-yl radical (1) was independently generated in single- and double-stranded RNA via photolysis of a ketone precursor. Direct stand breaks result from heterolytic cleavage of the adjacent C3'-carbon-oxygen bond. Trapping of 1 by O2 or ß-mercaptoethanol (1 M) does not compete with strand scission, indicating that phosphate elimination is >10(6) s(-1). Uracil loss also does not compete with strand scission. When considered in conjunction with reports that nucleobase radicals produce 1, this chemistry explains why RNA is significantly more susceptible to strand scission by ionizing radiation (hydroxyl radical) than is DNA.

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

Light-Triggered RNA Annealing by an RNA Chaperone.

Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.

Modified bleomycin disaccharides exhibiting improved tumor cell targeting.

The bleomycins (BLMs) are a family of antitumor antibiotics used clinically for anticancer chemotherapy. Their antitumor selectivity derives at least in part from their ability to target tumor cells, a property that resides in the carbohydrate moiety of the antitumor agent. In earlier studies, we have demonstrated that the tumor cell selectivity resides in the mannose carbamoyl moiety of the BLM saccharide and that both the BLM disaccharide and monosaccharide containing the carbamoyl moiety were capable of the delivery/uptake of a conjugated cyanine dye into cultured cancer cell lines. Presently, the nature of the participation of the carbamoyl moiety has been explored further to provide compounds of utility for defining the nature of the mechanism of tumor cell recognition and uptake by BLM saccharides and in the hope that more efficient compounds could be identified. A library of seven disaccharide-Cy5** dye conjugates was prepared that are structural analogues of the BLM disaccharide. These differed from the natural BLM disaccharide in the position, orientation, and substitution of the carbamoyl group. Studies of these compounds in four matched sets of tumor and normal cell lines revealed a few that were both tumor cell selective and internalized 2-4-fold more efficiently than the natural BLM disaccharide.

The disaccharide moiety of bleomycin facilitates uptake by cancer cells.

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

Independent generation and reactivity of uridin-2'-yl radical.

The uridin-2'-yl radical (1) has been proposed as an intermediate during RNA oxidation. However, its reactivity has not been thoroughly studied due to the complex conditions under which it is typically generated. The uridin-2'-yl radical was independently generated from a benzyl ketone (2a) via Norrish type I photocleavage upon irradiation at λmax = 350 nm. Dioxygen and ß-mercaptoethanol are unable to compete with loss of uracil from 1 in phosphate buffer. Thiol trapping competes with uracil fragmentation in less polar solvent conditions. This is ascribed mostly to a reduction in the rate constant for uracil elimination in the less polar solvent. Hydrogen atom transfer to 1 from ß-mercaptoethanol occurs exclusively from the α-face to produce arabinouridine. Mass balances range from 72 to 95%. Furthermore, the synthesis of 2a is amenable to formation of the requisite phosphoramidite for solid-phase oligonucleotide synthesis. This and the fidelity with which the urdin-2'-yl radical is generated from 2a suggest that this precursor should be useful for studying the radical's reactivity in synthetic oligonucleotides.

Selective tumor cell targeting by the disaccharide moiety of bleomycin.

In a recent study, the well-documented tumor targeting properties of the antitumor agent bleomycin (BLM) were studied in cell culture using microbubbles that had been derivatized with multiple copies of BLM. It was shown that BLM selectively targeted MCF-7 human breast carcinoma cells but not the "normal" breast cell line MCF-10A. Furthermore, it was found that the BLM analogue deglycobleomycin, which lacks the disaccharide moiety of BLM, did not target either cell line, indicating that the BLM disaccharide moiety is necessary for tumor selectivity. Not resolved in the earlier study were the issues of whether the BLM disaccharide moiety alone is sufficient for tumor cell targeting and the possible cellular uptake of the disaccharide. In the present study, we conjugated BLM, deglycoBLM, and BLM disaccharide to the cyanine dye Cy5**. It was found that the BLM and BLM disaccharide conjugates, but not the deglycoBLM conjugate, bound selectively to MCF-7 cells and were internalized. The same was also true for the prostate cancer cell line DU-145 (but not for normal PZ-HPV-7 prostate cells) and for the pancreatic cancer cell line BxPC-3 (but not for normal SVR A221a pancreas cells). The targeting efficiency of the disaccharide was only slightly less than that of BLM in MCF-7 and DU-145 cells and comparable to that of BLM in BxPC-3 cells. These results establish that the BLM disaccharide is both necessary and sufficient for tumor cell targeting, a finding with obvious implications for the design of novel tumor imaging and therapeutic agents.

Critical Review on Balanites aegyptiaca Delile: Phytoconstituents, Pharmacological Properties and Nanointerventions.

Balanites aegyptiaca Delile (BA) is an enduring xerophytic woody and spinous flowering tree and is commonly known as desert date or Ingudi (Hingot). It belongs to the family Zygophyllaceae, which is specific to be drought areas of Nigeria, Africa, South Asia and India (Rajasthan). In Ayurveda, this traditional medicinal plant is reported for the management of jaundice, syphilis, yellow fever, metabolic disorders, liver, and spleen problems. The main aim of the review is to compile its medicinal uses and further advancements to showcase the promises inherited in various parts of the plant for the benefit of mankind. As per the literature survey, various researchers have focused on the detailed investigation of BA including the phytopharmacological evidence, chemical constituents, nano-formulations, commercialized products, and clinical trials. Several remarkable scaffolds and isolated compounds like diosgenin, yamogenin, balanitin1/2, balanitin 3, bal4/5, bal6/7, rutin-3-glycosides, 3,7-diglycosides, (3, 12, 14, 16)-(12-hydroxycholest-5-ene-3,16-diyl-bis)-D-glucopyranoside and balanitoside have been identified. Additionally, this traditional plant has been scientifically proven by in vitro and in vivo. Based on the complete review of this plant, most of the compounds have been isolated from the fruit and kernel part. Additionally, based on the literature, a histogram was developed for pharmacological activity in which antidiabetic study was found to be more compared to other pharmacological activity. As a spinous desert dates, this plant needs to be explored more to bring out newer phytochemicals in the management of various diseases.