Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization.

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes .

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Synthesis of C3-Neoglycosides of digoxigenin and their anticancer activities.

Cardiac glycosides exhibit significant anticancer effects and the glycosyl substitution at C3 position of digoxigenin is pivotal for their biological activity. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and explore more potent anticancer agents, a series of C3-O-neoglycosides and C3-MeON-neoglycosides of digoxigenin were synthesized by the Koenigs-Knorr and neoglycosylation method, respectively. In addition, digoxigenin bisdigitoxoside and monodigitoxoside were prepared from digoxin by sodium periodate (NaIO4) oxidation and 6-aminocaproic acid hydrolysis. The SAR analysis revealed that C3-O-neoglycosides of digoxigenin exhibited stronger cytotoxicity and induction of Nur77 expression of tumor cells than C3-MeON-neoglycosides. Also, 3ß-O-glycosides exhibited stronger anticancer effects than 3α-O-glycosides. Among them, 3ß-O-(ß-l-fucopyranosyl)-digoxigenin (3i) showed the highest activity on induction of Nur77 expression and translocation from the nucleus to cytoplasm, leading to cancer cell apoptosis.

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications.

Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

Synthesis of MeON-neoglycosides of digoxigenin with 6-deoxy- and 2,6-dideoxy-d-glucose derivatives and their anticancer activity.

Cardiac glycosides show anticancer activities and their deoxy-sugar chains are vital for their anticancer effects. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and get more potent anticancer agents, a series of MeON-neoglycosides of digoxigenin was synthesized and evaluated. First, ten 6-deoxy- and 2,6-dideoxy-d-glucopyranosyl donors were synthesized starting from methyl α-d-glucopyranoside and 2-deoxy-d-glucose. Meanwhile, the digoxigenin was obtained by acidic hydrolysis of commercially available digoxin as glycosyl acceptor. Then, a 22-member MeON-neoglycoside library of digoxigenin was successfully synthesized by neoglycosylation method. Finally, the induction of Nur77 expression and its translocation from the nucleus to cytoplasm together with cytotoxicity of these MeON-neoglycosides were evaluated. The SAR analysis revealed that C3 glycosylation is required for their induction of Nur77 expression. Moreover, some MeON-neoglycosides (2b and 8b) could significant induce the expression of Nur77 and its translocation from the nucleus to cytoplasm. However, these compounds showed no inhibitory effects on the proliferation of cancer cells, suggesting that they may not induce apoptosis of NIH-H460 cancer cells and their underlying potential and application toward cancer cells deserves future study.

Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity.

Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.

Analysis of RNA transport in Xenopus oocytes and mammalian cells.

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.

Computational design of ligand-binding proteins with high affinity and selectivity.

The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and ß-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.

PK modulation of haptenylated peptides via non-covalent antibody complexation.

We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide-IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide-antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody-hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.

Probing the regiospecificity of enzyme-catalyzed steroid glycosylation.

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-ß-D-glucoside.

Steered molecular dynamics simulations of ligand-receptor interaction in lipocalins.

Retinol binding protein (RBP) and an engineered lipocalin, DigA16, have been studied using molecular dynamics simulations. Special emphasis has been placed on explaining the ligand-receptor interaction in RBP-retinol and DigA16-digoxigenin complexes, and steered molecular dynamics simulations of 10-20 ns have been carried out for the ligand expulsion process. Digoxigenin is bound deep inside the cavity of DigA16 and forms several stable hydrogen bonds in addition to the hydrophobic van der Waals interaction with the aromatic side-chains. Four crystalline water molecules inside the ligand-binding cavity remain trapped during the simulations. The strongly hydrophobic receptor site of RBP differs considerably from DigA16, and the main source of ligand attraction comes from the phenyl side-chains. The hydrogen bonds between digoxigenin and DigA16 cause the rupture forces on ligand removal in DigA16 and RBP to differ. The mutated DigA16 residues contribute approximately one-half of the digoxigenin interaction energy with DigA16 and, of these, the energetically most important are residues His35, Arg58, Ser87, Tyr88, and Phe114. Potential "sensor loops" were found for both receptors. These are the outlier loops between residues 114-121 and 63-67 for DigA16 and RBP, respectively, and they are located near the entrance of the ligand-binding cavity. Especially, the residues Glu119 (DigA16) and Leu64 (RBP) are critical for sensing. The ligand binding energies have been estimated based on the linear response approximation of binding affinity by using a previous parametrization for retinoids and RBP.

A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing.

SUMMARY: A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.

DNA aptamer folding on magnetic beads for sequential detection of adenosine and cocaine by substrate-resolved chemiluminescence technology.

We have developed a new aptamer-based chemiluminescence (CL) biosensing platform for the sequential detection of two small molecules as exemplified by adenosine and cocaine. Each biosensing platform comprises NH(2)-functionalized capture DNA immobilized on magnetic beads; this can hybridize with one end of the aptamer. A corresponding reporter DNA probe labeled with either digoxigenin or biotin on its 5'-terminus recognizes the other end of the aptamer. The target compounds adenosine or cocaine act as specific competitors to aptamer-reporter DNA binding, and the corresponding aptamers preferentially form target-aptamer complexes. This results in detachment of the reporter DNA probe from the magnetic beads, with more target molecules resulting in less reporter DNA probe remaining on the beads. Those left are sequentially detected by using substrate-resolved anti-digoxigenin-alkaline phosphatase and streptavidin-horseradish peroxidase. Experimental results confirm that this CL immunosensing platform has good sensitivity with detection limits of 5.2 x 10(-9) M and 3.2 x 10(-9) M for adenosine and cocaine, respectively. Because it is straightforward to adapt this strategy to detect a spectrum of small molecules by using different aptamers, this method may offer a new direction in designing high-performance CL aptasensors for sensitive and sequential determination of a limited number of small molecules.

COMU: a safer and more effective replacement for benzotriazole-based uronium coupling reagents.

We describe a new family of uronium-type coupling reagents that differ in their iminium moieties and leaving groups. The presence of the morpholino group in conjunction with an oxime derivative--especially ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma)--had a marked influence on the solubilities, stabilities, and reactivities of the reagents. Finally, the new uronium salt derived from Oxyma (COMU) performed extremely well in the presence of only 1 equiv of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. COMU also showed a less hazardous safety profile than the benzotriazole-based HDMA and HDMB, which exhibited unpredictable autocatalytic decompositions. Furthermore, the Oxyma moiety contained in COMU suggests a lower risk of explosion than in the case of the benzotriazole derivatives.

Digoxigenin modification of adenovirus to spatially control gene delivery from chitosan surfaces.

To spatially control the delivery of multiple viral vectors from biomaterial scaffolds, digoxigenin (DIG) was conjugated to adenoviral capsid proteins as an antigenic determinant for antibody immobilization. The infectivity, toxicity, specificity and immobilization stability of DIG-modified adenovirus were examined to investigate the feasibility and effectiveness of this viral surface modification. Anti-DIG antibody conjugated on chitosan surfaces was able to immobilize DIG-modified adenovirus and could be stably bound on the material for at least two weeks, yet the modification was mild enough that viral infectivity was maintained. To immobilize two different adenoviruses, wax masking was applied to conjugate anti-DIG and anti-adenovirus antibodies in two discrete regions of a chitosan film. The distribution of these two viral vectors expressing different reporter genes was examined after cell culture. Fluorescent protein expression from transduced cells illustrated that the infection distribution could be controlled: one gene was delivered to the entire region of the biomaterial, and another was only delivered to defined regions. Compared to three other cardiac glycosides, ATPase inhibition was undetectable when DIG was conjugated on the adenovirus, suggesting that the method may be safe for in vivo application. This dual viral vector delivery system should be capable of generating distinct interfaces between cell signaling viruses to control tissue regeneration from a range of different biomaterials.

Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5.

The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans--one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing beta(1-4)-N-acetyl-D-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galbeta(1-4)-GlcNAc(x) component found in other Pgps.

[A new method for EMSA by modifying DIG high prime DNA labeling and detection starter Kit II].

Gel retardation, also named electrophoretic mobility shift assay (EMSA), is a useful tool for identifying protein-DNA interactions. Typically, 32P-labeled DNA probes used in EMSA are sensitive. However, it relies on the handling of hazardous radioisotopes, and is not easily quantified. Recently, some successful cases have been reported using non-radio labelled probes instead of radiolabelled probes in EMSA. The method is rapid, convenient, and safe, but it depends on a very expensive kit. In this study, we offered a new method performing EMSA by modifying DIG High Prime DNA Labeling and Detection Starter Kit II (Rohe). Firstly, the prepared labeled probe was introduced the EcoR I stick in the end of probe for 3'-end labeling, and then was performed the probe labeling and detecting the signals of EMSA with the relatively cheap DIG High Prime DNA Labeling and Detection Starter Kit II Rohe. By adjusting the experiment parameters, the successful result was obtained. The present study provides a successful example and method for modifying DIG High Prime DNA Labeling and Detection Starter Kit II.

Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer.

Early detection is the hallmark of successful cancer treatment. Evidence is accumulating that primary cancers begin shedding neoplastic cells in the circulation at an early stage. To date, a high-sensitivity and high-throughput method for the detection of circulating tumor cells (CTCs) is deficient. In this study, we have developed a high-sensitivity colorimetric membrane-array method to detect CTCs in the peripheral blood of colorectal cancer (CRC) patients as a potential diagnostic tool. Previously, we identified a set of 18 oligonucleotide clones, significantly overexpressed in CRC, which were synthesized and applied to a nylon membrane. Digoxigenin (DIG)-labeled cDNA were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the peripheral blood of 88 Taiwanese CRC patients and 50 healthy subjects, and were then hybridized to the membrane-array. Hybridization signals were detected by color development. Meanwhile, blood samples were analyzed by real-time quantitative PCR (Q-PCR). Subsequently, both methods were compared regarding their correlation, sensitivity and specificity in the detection of CTCs by statistics. The results of membrane-arrays were demonstrated to be closely related to that of Q-PCR (P<0.001). The sensitivity and specificity of membrane-arrays for the detection of CTCs were 94.3% (95% CI, 86.4-102.2%) and 94% (95% CI, 85.9-102.1%), respectively. Moreover, the accuracy of membrane-arrays is higher than that of any one gene by Q-PCR. The detection rate of membrane-arrays was significantly associated with the depth of tumor invasion (P=0.002), the presence of lymph node metastasis (P=0.016), and TNM stage (P=0.005). The preliminary results indicated that the accuracy of membrane-arrays was sufficient to distinguish Taiwanese CRC patients from normal individuals with the advantages of time-saving, cost-effectiveness and high-throughput. Thus, the constructed colorimetric membrane-array could be a promising approach for the future detection of CTCs.