Improvement of a miRNA inhibitor by intracellular selection.

Sequences surrounding the miRNA binding domain of the miRNA inhibitor LidNA were selected intracellularly. The library was transfected into cells, and then, inhibitors that were associated with argonaute 2 were selected. The potent inhibitors were slowly degraded intracellularly, while the lower-activity inhibitors were rapidly degraded. A combination of the selected sequences surrounding the miRNA binding domain enhanced miRNA inhibitory activity. ABBREVIATIONS: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; Ago2: argonaute 2; LNA: locked nucleic acid.

Exploiting the biological efficacy of benzimidazole based Schiff base complexes with l-Histidine as a co-ligand: Combined molecular docking, DNA interaction, antimicrobial and cytotoxic studies.

Four new metal complexes were synthesized and screened for their cytotoxic activity after sufficient assertion from the preliminary DNA binding studies. The metal complexes could bind to CT-DNA through intercalation binding mode. This has also been confirmed by the molecular docking studies. The DNA cleavage efficiencies of these complexes with pBR322 DNA were investigated by gel electrophoresis. The complexes were found to promote the cleavage of pBR322 DNA from the supercoiled form I to the open circular form II in the presence of an oxidizing agent (H2O2). The in vitro chemosensitivity of the studied complexes exhibits significant cytotoxic effects, compared to those reported for cisplatin. These findings represent a prompting to search for the probable interaction of these complexes with other cellular elements of fundamental consequence in cell proliferation. The in vitro anticancer activities indicate that the Cu(II) complex is active against the selected human tumor cell lines, and the order of in vitro anticancer activities is consistent with the DNA-binding affinities. Towards noncancerous cell line, Cu(II) complex exhibits very low toxicity. On the other hand all the complexes have been found to exhibit cytotoxic effects against cancerous cell lines with potency more than that of the widely used drug cisplatin and hence they have the potential to act as promising anticancer agents. Captivatingly, they are non-toxic to normal cell lymphocytes revealing that they are selective in killing only the cancer cells.

A metal-organic framework based PCR-free biosensor for the detection of gastric cancer associated microRNAs.

We report herein five sensing platforms for the detection of five gastric cancer associated microRNAs (miRNAs). The sensing platforms are hybrids formed from a water-stable metal organic framework (MOF) {[Cu(dcbb)2(H2O)2]·10H2O}n (1, H2dcbbBr=1-(3,5-dicarboxybenzyl)-4,4'-bipyridinium bromide), respectively with five carboxyfluorescein (FAM) labeled probe single-stranded DNA (probe ss-DNA, denoted as P-DNA). Within the hybrid, MOF 1 tightly interacts with the P-DNA through electrostatic and/or π-stacking interactions and results in fluorescence quenching of FAM via a photo-induced electron transfer (PET) process. In the presence of the complementary target miRNAs miR-185, miR-20a, miR-92b, miR-25 and miR-210, which are expressed abnormally in the plasma of gastric carcinoma patients, P-DNA is released from the surface of MOF 1 ascribed to the stronger base pair matching, leading to the FAM fluorescence recovery. Each P-DNA@1 system is effective and reliable for the detection of its complementary target miRNA with the detection limits from 91 to 559pM, and is not interfered by other four miRNA sequences.

DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes.

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

Nuclear Localization and Gene Expression Modulation by a Fluorescent Sequence-Selective p-Anisyl-benzimidazolecarboxamido Imidazole-Pyrrole Polyamide.

Synthetic pyrrole (P)-imidazole (I) containing polyamides can target predetermined DNA sequences and modulate gene expression by interfering with transcription factor binding. We have previously shown that rationally designed polyamides targeting the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα (topo IIα) promoter can inhibit binding of transcription factor NF-Y, re-inducing expression of the enzyme in confluent cells. Here, the A/T recognizing fluorophore, p-anisylbenzimidazolecarboxamido (Hx) was incorporated into the hybrid polyamide HxIP, which fluoresces upon binding to DNA, providing an intrinsic probe to monitor cellular uptake. HxIP targets the 5'-TACGAT-3' sequence of the 5' flank of ICB2 with high affinity and sequence specificity, eliciting an ICB2-selective inhibition/displacement of NF-Y. HxIP is readily taken up by NIH3T3 and A549 cells, and detected in the nucleus within minutes. Exposure to the polyamide at confluence resulted in a dose-dependent upregulation of topo IIα expression and enhanced formation of etoposide-induced DNA strand breaks.

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides.

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.

Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe.

The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC(200) of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.

Potency and property of DNA conformational damage determine ATM activation: two new DNA topological probes.

We have shown previously the intercalation geometry of a series of acenaphtho [1,2-b] pyrrole derivatives with DNA double helix in vitro. In this report we chose a couple of intercalating analogues and a Chinese traditional medicine Tanshinone IIA as probes to investigate the response of DNA damage sensor ataxia-telangiectasia mutated (ATM) protein toward the DNA topological change in vivo. The two analogues (1)a (3-(4-Methyl-piperazin)-8-oxo-8H-acenaphtho [1,2-b]pyrrole -9-carbonitrile) and (3)a (3-(3-Dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9- carbonitrile) could unwind double helix to different extents, whereas Tanshinone IIA could wind the double helix. Using a combination of circular dichroism (CD) studies and immunoflurescence assays, we found for the first time that the ATM protein kinase can respond to the unwinding chromatin conformational damage caused by (1)a and (3)a, while it could not be activated by the winding effects caused by Tanshinone IIA. Moreover, the amount of ATM protein phosphorylation is consistent with the degree of unwinding conformational damage. The average number of ATM foci in an MCF-7 cell is 32 +/- 1.5 at 6 microM (1)a, which is significantly higher than the 8 microM (3)a exposure (15 +/- 0.5, p < 0.5). A new couple of DNA topological probes, (1)a and (3)a have been found for the future semi-quantitative investigation of factors involved in the DNA damage pathway.

Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification Process.

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.

Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates.

Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)(8) (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra.

Improved DNA probe detection of Listeria monocytogenes in enrichment culture after physical-chemical fractionation.

Bacterial detection in foods by nucleic acid probes is limited by microflora competition during selective enrichment. Probe target concentration by extraction and fractionation of enrichments may diminish this limitation. The 1-h AccuProbe chemiluminescent culture identification test for Listeria monocytogenes was used as a model. Its high detection threshold provides a stringent challenge for evaluating enrichment work-up protocols. Detection of L. monocytogenes, at 1-4 colony-forming units/g food, was not consistently possible in 48 h enrichment cultures using AccuProbe. Concentration by cell sedimentation was occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sediments were sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNA was separated from non-RNA material by extraction with phenol and precipitation with ethanol. Enrichments (250 mL) were concentrated 2500-fold, and the limitation was food RNA volume. A strongly competitive Enterococcus faecium food isolate was used to demonstrate the effect of artificial competition on the kit's ability to detect L. monocytogenes in enrichments. High competitor concentrations repressed the level of the target below the detection threshold, but concentration of r-RNA enabled detection of L. monocytogenes. The effectiveness of this enrichment sample work-up was demonstrated with naturally contaminated hummus.

Controlled expression of an rpoS antisense RNA can inhibit RpoS function in Escherichia coli.

We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents.

Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells.

In this study, the interrelationship between signal transduction pathways and 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] action was examined in UMR106 osteoblastic cells. Treatment of these cells with 8-bromo-cAMP (1 mM) resulted in an upregulation of the vitamin D receptor (VDR) and an augmentation in the induction by 1,25(OH)2D3 of 25(OH)D3 24-hydroxylase [24(OH)ase] and osteopontin (OPN) mRNAs as well as gene transcription. Transfection with constructs containing the vitamin D response element devoid of other promoter regulatory elements did not alter the cAMP-mediated potentiation, suggesting that cAMP-enhanced transcription is due, at least in part, to upregulation of VDR. Treatment with phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA) 100 nM], an activator of protein kinase C, significantly enhanced 1,25(OH)2D3-induced OPN mRNA and transcription but had no effect on VDR or on 24(OH)ase mRNA or transcription. Studies using OPN promoter constructs indicate that TPA-enhanced OPN transcription is mediated by an effect on the OPN promoter separate from an effect on VDR. Thus interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, changes in cellular phosphorylation may play a significant role in determining the effectiveness of 1,25(OH)2D3 on transcriptional control in cells expressing skeletal phenotypic properties.

Interaction of nuclear proteins with repeat sequences in the 5' flanking region of mouse muscle creatine kinase gene during aging.

The 5' flanking region of the mouse muscle creatine kinase (MCK) gene contains two repeat sequences-a mononucleotide repeat, (A)(22) (-2694 to -2673), and a tetranucleotide repeat, (GTTT)(8) (-2962 to -2931). We show here that these repeats in the mouse MCK gene bind to specific nuclear protein factors. Some of the factors interacting with these sequences are tissue-specific and show age-related decrease in the binding activity. Nonspecific competitor and heterologous DNA probes failed to compete out the complexes showing that the interaction is specific to the repeat sequences. These proteins may have a role in the expression of the gene during aging.

Modulation of RNase H activity by modified DNA probes: major groove vs minor groove effects.

We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex. The duplexes were treated with Escherichia coli RNase H. Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes. However, minor groove substituents inhibited RNA cleavage over a four-base region. Comparison was made with a DNA probe containing a 2'-OMe modification. Our results support enzyme binding in the minor groove of a DNA/RNA duplex. We do not observe cleavage directly across from the modified nucleoside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA. Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target. The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.

The newborn rabbit sino-atrial node expresses a neuronal type I-like Na+ channel.

1. Newborn rabbit sino-atrial node (SAN) myocytes were recently found to express a tetrodotoxin (TTX)-sensitive Na+ current. We now report that the dose-response relation indicates that this SAN Na+ channel has unusually high TTX sensitivity, with half-maximal inhibition (26 +/- 5 nM) which is more typical of neuronal than cardiac tissue. 2. Additional characterization used mu-conotoxin GIIIA and Cd2+ as relatively selective blockers of the skeletal and cardiac isoforms, respectively. mu-Conotoxin GIIIA had no effect on the current recorded from SAN myocytes, but the Cd2+ sensitivity was unexpectedly high for a neuronal isoform (half-maximal inhibition = 185 +/- 8 microM). 3. Analysis of the time constant of inactivation did not reveal evidence of multiple inactivation processes, with the data well fitted by a single, relatively rapid exponential (inactivation time constant = 0.58 +/- 0.03 ms at 0 mV). 4. In situ hybridization with anti-sense cDNA probes was used to test for expression of neuronal type I, II and III Na+ channel isoforms. Myocardial cells in newborn SAN tissue exhibited clear hybridization to the type I, but not the type II or III probes. No hybridization was observed in adult SAN tissue with any of the three probes. 5. It is concluded that the newborn SAN expresses a neuronal type I-like Na+ channel isoform, and that this probably accounts for the unusual characteristic of high sensitivity to both TTX and Cd2+.

Interaction of tetra-azaphenanthrene ruthenium complexes with DNA and oligonucleotides. A photophysical and photochemical investigation.

The interaction of complexes Ru(bpy)n(TAP)3-n2+ (1a-1d for n = 0-3) with DNA has been investigated using photophysical methods (emission spectroscopy and laser flash photolysis), by studying the induction of single-strand breaks in plasmid DNA and the formation of adducts using 32P-labelled 27-mer oligonucleotides. Two classes of behaviour are found. Complexes 1a and 1b show quenched emission in the presence of calf thymus DNA and yield photoadducts with the 27-mer, whereas 1c and 1d show enhanced emission and do not form photoadducts. 1a and 1b are more efficient sensitizers for single-strand breaks than are 1c and 1d. It is proposed that the excited states of 1a and 1b, which are stronger oxidizing agents than those of 1c and 1d, are capable of oxidizing guanine. Direct evidence for electron transfer has been obtained from laser flash photolysis of Ru(TAP)3(2+) and dGMP, CT-DNA and polynucleotides.

Uranyl photoprobing of conformational changes in DNA induced by drug binding.

The effect on DNA conformation upon binding of the bis-intercalator, N,N'-diacridinyl spermidine is studied by uranyl mediated DNA photocleavage and gel retardation. It is shown that a characteristic A-tract correlated 10 base pair modulation of the uranyl photocleavage of bent (kinetoplast) DNA is suppressed upon binding of the diacridine. Likewise, the anomalous slow gel electrophoretic migration of bent-DNA is abolished by diacridine binding. At higher diacridine/DNA ratios a DNA conformation in which G-residues become hypersensitive to uranyl photocleavage is induced.