Excitonically Coupled Cyclic BF2 Arrays of Calix[8]- and Calix[16]phyrin as Near-IR-Chromophores.

Two giant calix[n]phyrin derivatives namely calix[8]- (4) and calix[16]phyrin (5), involving two and four BF2 units, respectively, were prepared through the condensation of the bis-naphthobipyrrolylmethene-BF2 complex (3) with pentafluorobenzaldehyde. Calix[n]phyrins 4 and 5 display extremely high extinction coefficients (3.67 and 4.82×105 m-1 cm-1 , respectively) in the near-IR region, which was taken as initial evidence for strong excitonic coupling within these cyclic multi-chromophoric systems. Detailed insights into the effect of excitonic coupling dynamics on the electronic structure and photophysical properties of the macrocycles came from fluorescence, time-correlated single-photon counting (TCSPC) and transient absorption (TA) measurements. Support for these experimental findings came from theoretical studies. Theory and experiment confirmed that the coupling between the excitons depends on the specifics of the calix[n]phyrin structure, not just its size.

Tetrazolylpyrene unnatural nucleoside as a human telomeric multimeric G-quadruplex selective switch-on fluorescent sensor.

We report herein the specific sensing of dimeric H45 G-quadruplex DNA via a fluorescence light-up response using fluorescent tetrazolylpyrene nucleoside (TzPyBDo) as a probe. The strong binding of the probe via an intercalative stacking interaction inside the connecting loop of two G-quadruplex units of H45 and the discrimination to other monomeric and long DNA duplexes are accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability.

Regioselective and stereoselective route to N2-ß-tetrazolyl unnatural nucleosides via SN2 reaction at the anomeric center of Hoffer's chlorosugar.

We are reporting a regioselective and stereoselective route to N2-ß-tetrazolyl aromatic donor/acceptor unnatural nucleosides as new class of possible DNA base analogs. The SN2 substitution reaction at the anomeric center of Hoffer's chlorosugar with various 5-substituted aromatic tetrazoles in THF in presence of K2CO3 proceeds with regioselectivity at N2-tetrazoles and stereoselectivity at α-chlorosugar with very good yield. The stereoelectronic and steric effects play a crucial role for the observed outcome which is also supported from a theoretical (DFT) study. The methodology is simple, eco-compatible and the tetrazolyl unnatural nucleosides might find applications in decorating DNA for various biotechnological and DNA based material science applications.

Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: synthesis, photophysical properties and study of interaction with BSA.

Much effort has been put forth to develop unnatural, stable, hydrophobic base pairs with orthogonal recognition properties and study their effect on DNA duplex stabilisation. Our continuous efforts on the design of fluorescent unnatural biomolecular building blocks lead us to the synthesis of some triazolyl donor/acceptor unnatural nucleosides via an azide-alkyne 1,3-dipolar cycloaddition reaction as a key step, which we want to report herein. We have studied their photophysical properties and found interesting solvatochromic fluorescence for two of the nucleosides. Photophysical interactions among two donor-acceptor ß-nucleosides as well as a pair of α/ß-nucleosides have also been evaluated. Furthermore, we have exploited one of the fluorescent nucleosides in studying its interaction with BSA with the help of UV-visible and steady state fluorescence techniques. Our design concept is based on the hypothesis that a pair of such donor/acceptor nucleosides might be involved in π-stacking as well as in photophysical interactions, leading to stabilization of the DNA duplex if such nucleosides can be incorporated into short oligonucleotide sequences. Therefore, the designed bases may find application in biophysical studies in the context of DNA.

Triazolyl donor/acceptor chromophore decorated unnatural nucleosides and oligonucleotides with duplex stability comparable to that of a natural adenine/thymine pair.

We report the design and synthesis of triazolyl donor/acceptor unnatural nucleosides via click chemistry and studies on the duplex stabilization of DNA containing two such new nucleosides. The observed duplex stabilization among the self-pair/heteropair has been found to be comparable to that of a natural A/T pair. Our observations on the comparable duplex stabilization has been explained on the basis of possible π-π stacking and/or charge transfer interactions between the pairing partners. The evidence of ground-state charge transfer complexation came from the UV-vis spectra and the static quenching of fluorescence in a heteropair. We have also exploited one of our unnatural DNAs in stabilizing abasic DNA.

Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency.

Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucine-zipper (bZIP) transcription factor gene family, bZIP19 and bZIP23, that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt- and Irt-related protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils.

Targeting Oncogene Promoters and Ribosomal RNA Biogenesis by G-Quadruplex Binding Ligands Translate to Anticancer Activity.

G-Quadruplex (GQ) nucleic acids are promising therapeutic targets in anticancer research due to their structural robustness, polymorphism, and gene-regulatory functions. Here, we presented the structure-activity relationship of carbazole-based monocyanine ligands using region-specific functionalization with benzothiazole (TCA and TCZ), lepidine (LCA and LCZ), and quinaldine (QCA and QCZ) acceptor moieties and evaluated their binding profiles with different oncogenic GQs. Their differential turn-on fluorescence emission upon GQ binding confirmed the GQ-to-duplex selectivity of all carbazole ligands, while the isothermal titration calorimetry results showed selective interactions of TCZ and TCA to c-MYC and BCL-2 GQs, respectively. The aldehyde group in TCA favors stacking interactions with the tetrad of BCL-2 GQ, whereas TCZ provides selective groove interactions with c-MYC GQ. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) showed that these molecules interfere with the recruitment of specific transcription factors at c-MYC and BCL-2 promoters and stabilize the promoter GQ structures to inhibit their constitutive transcription in cancer cells. Their intrinsic turn-on fluorescence response with longer lifetimes upon GQ binding allowed real-time visualization of GQ structures at subcellular compartments. Confocal microscopy revealed the uptake of these ligands in the nucleoli, resulting in nucleolar stress. ChIP studies further confirmed the inhibition of Nucleolin occupancy at multiple GQ-enriched regions of ribosomal DNA (rDNA) promoters, which arrested rRNA biogenesis. Therefore, carbazole ligands act as the "double-edged swords" to arrest c-MYC and BCL-2 overexpression as well as rRNA biogenesis, triggering synergistic inhibition of multiple oncogenic pathways and apoptosis in cancer cells.

Metal catalyst-free photo-induced alkyl C-O bond borylation.

Metal catalyst free, blue visible light-induced C-O bond borylation of unactivated tertiary alkyl methyl oxalates has been developed to furnish tertiary alkyl boronates. From the secondary alcohols activated with imidazolylthionyl, moderate yields of boronates were attained under standard photo-induced conditions. Preliminary mechanistic studies confirmed the involvement of a (DMF)2-B2cat2 adduct that weakly absorbs light at 437 nm so as to initiate a Bcat radical. A radical-chain process is proposed wherein the alkyl radical is engaged.

Stabilization of an abasic site paired against an unnatural triazolyl nitrobenzene nucleoside.

An abasic site is the most frequently observed among the various forms of DNA lesions in genomic DNA. If left unrepaired, an abasic site might turn out to be a principle cause for deleterious mutations and can be threat to cellular survival. Thus, to keep cellular integrity and measure the extent of DNA damage, recognition and stabilization of the abasic sites (apurinic/apyrimidinic site = Ap) are essential. Further, it is crucial to detect and stabilise the abasic site for towards the development of new diagnostics and chemotherapeutics. Herein, we report the stabilization of an abasic DNA duplex wherein the abasic site paired against a novel unnatural nucleoside, triazolylnitrobenzene (TNBBAc). This nucleoside is bulky and exhibits, high polarizability and good stacking propensity. Robust hetero-pair stabilization is another feature of it. Therefore, it is interesting to study the stabilization of an abasic DNA containing a synthesized triazolylnitrobenzene nucleoside TNBBAc We planned to study the thermal as well as the thermodynamic origin of abasic DNA stabilization by our synthesized oligonucleotide probe containing TNBBAc nucleoside. We observed that the nucleoside TNBBAc offered good thermal stabilization of a TNBBAc-Φ duplex via strong intercalative stacking interaction alongside an abasic site. The UV-visible spectroscopic study supported the intercalative stacking interaction. The stabilization though is marginal, but it would shed light on the design of bases of significant volume to stabilise abasic DNA to a greater extent.

Trifunctional fluorescent unnatural nucleoside: Label free detection of T-T/C-C base mismatches, abasic site and bulge DNA.

The detection and targeting of both the mismatched and abasic DNA is highly important which would ultimately help in designing new diagnostics and chemotherapeutics. Furthermore, sensing and targeting the bulge sequence with a fluorescent probe would be useful to study the role of bulges in nucleic acid function or could have significant therapeutic potential. Thus, detection of specific bulges by small fluorescent molecules is an attractive research area since the past several years. Many attempts have been made to prepare such compounds. We report herein a label free strategy for the detection of pyrimidine base mismatches (T/T and C/C), sensing of abasic site, and pyrimidine base bulge DNA using an unnatural tetrazolylpyrene nucleoside (TPyBDo) as a bare fluorescent probe. The H-bonding/hydrophobic force mediated interactions allow the sensing of all three deformed DNA via an enhancement of fluorescence signal using our simple "Just-Mix and Read" strategy. The binding of the probe to all the three deformed DNA duplexes is accompanied by an increase in the thermal melting stability of the deformed DNAs. That the probe binds efficiently to the minor groove near the deformed site was evident from spectroscopic studies. All the spectral evidences open up a multitude of possibilities for using our probe, tetrazolylpyrene nucleoside, as an efficient fluorescent light-up bio-probe for label free DNA detection.

Expression of the ZNT1 Zinc Transporter from the Metal Hyperaccumulator Noccaea caerulescens Confers Enhanced Zinc and Cadmium Tolerance and Accumulation to Arabidopsis thaliana.

Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5' deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading.

Bioinformatic approaches for identification and characterization of olfactomedin related genes with a potential role in pathogenesis of ocular disorders.

PURPOSE: To identify olfactomedin domain containing proteins, which are expressed in the eye and have similarity to myocilin, to test as potential candidates for eye diseases. Most of the mutations in myocilin causing primary open angle glaucoma are located in the olfactomedin domain. In vitro experiments demonstrated interaction between optimedin and myocilin through the conserved olfactomedin domains of the proteins in rats, and it was speculated that optimedin might have a role in the pathogenesis of ocular disorders. Hence, we aimed to identify myocilin related human proteins having conserved olfactomedin domains with potential to interact between them and examine the expression patterns in the eye by bioinformatics approaches. This endeavor would have the potential to identify new candidate genes for eye diseases in general and glaucoma in particular to be tested by wet-lab experiments. METHODS: Proteins with homology to myocilin were selected by BLASTp at the NCBI server. cDNA sequences and corresponding genomic contigs were retrieved. Pairwise BLAST was done to investigate the gene structure. The human EST database and NEIBank were searched against the selected cDNAs to look for tissue specific expression of the transcripts. RESULTS: The study led to the identification of three groups of proteins encoded by three different genes; Noelin 1 (9q34.3), Noelin 2 (19p13.2), and Noelin 3 (1p22) encompassing 45,575 bp, 82,679 bp, and 1,93,421 bp of the genomic sequence, respectively. Genomic structures, alternate usage of exons, and molecular evolution of the Noelins were determined. Similar structures of the genes, splicing patterns and high levels of homology shed light on the relatedness and molecular evolution of this group of olfactomedin related proteins. Strikingly, however, Noelin 1 and Noelin 3 were found to be expressed as multiple splice variants while only a single spliced transcript could be identified for Noelin 2. A human EST database search suggested the expression of all three Noelin genes in the brain but only two (Noelin 1 and Noelin 2) in the eye despite experimental evidence for expression of Noelin 3 in ocular tissue. Myocilin was determined to have similar levels (60-61%) of homology with all three Noelin gene products (Noelin 1_v1, Noelin 2_v1, and Noelin 3_v1) at the conserved olfactomedin domains. CONCLUSIONS: Mammalian Noelin 1 evolved from its precursor, followed by evolution of Noelin 3 and Noelin 2 by gene duplication events. Myocilin might have evolved from Noelin 2 by gene duplication followed by exon fusion. Noelin 1 and Noelin 2 could be tested as candidate genes for eye diseases based on their expressions in the eye and shared olfactomedin domains with Myocilin in the C-termini of the respective proteins.

Design and synthesis of triazolyl-donor/acceptor unnatural nucleosides and oligonucleotide probes containing triazolyl-phenanthrene nucleoside.

In the context of abasic DNA or DNA duplex stabilization, several unnatural nucleosidic/non-nucleosidic base surrogates have been reported. Toward this end, we have designed and synthesized triazolyl-aromatic donor chomophores as unnatural nucleoside analogs. These modifications display markedly higher thermal stabilization of abasic DNA duplex in comparison to the stabilization offered by other nucleoside/non-nucleoside base surrogates reported in the literature. The same oligonucleotide probe containing triazolylphenanthrene nucleotide also offers very good stability of the self-pair duplex via π-π stacking interaction and hetero-pair duplex via charge transfer interaction when paired against triazolyl acceptor aromatic nucleoside. Moreover, the probe in the reverse sequence containing triazolylphenanthrene nucleotide has shown FRET efficiency in a chimeric DNA duplex. The triazolyl nucleotides would expectedly show stability toward exonuclease activity. This unit describes protocols for chemical synthesis of unnatural triazolyl nucleosides and one oligonucleotide probe. The unit also provides a summary of various thermal and photophysical applications of triazolylphenantherene-containing oligonucleotides.

Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair.

Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate () is paired against a fluorescent nucleosidic base surrogate (). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor or direct excitation of the FRET acceptor .