Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions.

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Northern Blot Detection of Tiny RNAs.

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).

Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

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 .

Application of Antibody-Powered Triplex-DNA Nanomachine to Electrochemiluminescence Biosensor for the Detection of Anti-Digoxigenin with Improved Sensitivity Versus Cycling Strand Displacement Reaction.

The accurate and rapid quantitative detection of antibodies had a significant influence in controlling and preventing disease or toxin outbreaks. In this work, we first introduce the antibody-powered triplex-DNA nanomachine to release cargo DNA as a substitute target for sensitive electrochemiluminescence (ECL) detection of anti-digoxigenin based on a novel ternary ECL system. It is worth noting that the cargo DNA as a substitute target of antibody can further participate in an enzyme-assisted cycling strand displacement reaction to achieve ECL signal amplification and improve the sensitivity of antibody detection. Additionally, porous palladium nanospheres with a considerable catalytic activity were first applied as a coreaction accelerator to efficiently enhance the intensity of the ECL system of rubrene microblocks as luminophore and dissolved O2 as an endogenous coreactant. With the resultant ternary ECL system as a biosensing platform, a significantly enhanced initial signal was achieved in advance. Then, the ferrocene-labeled quenching probes were employed to reduce initial signal and obtain the low-background signal. Eventually, the cargo DNA made the quenching probes release and recover the signal in the presence of anti-digoxigenin. Thereupon, the wide linear range (0.01-200 nM) and low limit of detection (6.7 pM) were obtained, and this method not only reduces conjugation steps but also provides a sensitive and novel ECL analysis platform for the trace detection of other antibodies and antigen.

Design of a stretchable DNAzyme for sensitive and multiplexed detection of antibodies.

Advanced methods for developing and applying responsive DNA nanodevices are of great interest. Herein, we report a stretchable DNAzyme that allows simple, multiplexed and sensitive fluorescent detection of antibodies. We find that rigid antibody can tightly stretch soft, antigen-labelled DNAzyme strand and disrupt the hybridization between DNAzyme and its substrate. Based on this finding, we develop a novel strategy to detect antibodies. Due to the robustness and high activity of DNAzyme, this assay can easily detect target as low as 1 ±â€¯0.25 pM and achieve multiplexed detection by using a cocktail of DNAzymes. The proposed assay not only provides a new approach to readily measure antibody, but broadens the application of DNAzyme that is usually employed to detect metal ions or indirectly analyze biomolecules without the cumbersome design.

Antibody-Bridged Beacon for Homogeneous Detection of Small Molecules.

In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences is required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmarks, e.g., binding energy of -11 kcal/mol, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated in the analyses of two small molecule targets, biotin and digoxin. Low nanomolar limits of detection were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.

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.

Nonradioactive Northern Blot of circRNAs.

Circular RNAs (circRNAs) are recognized as a special species of transcripts in metazoans with increasing studies, and northern blotting is a direct way to confirm the existence and to evaluate the size of individual circRNAs. Northern blotting probes can be radioactive isotope (32P) labeled, which is not environment-friendly and sometimes inconvenient to use. Here, we describe a nonradioactive northern blot protocol with digoxigenin-labeled probe to detect circRNA.

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.

Improved Free-Energy Landscape Quantification Illustrated with a Computationally Designed Protein-Ligand Interaction.

Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (koff ) and the distance to the transition state (Δx≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG≠ =6.3±0.2 kcal mol-1 ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [koff (=4±0.1×10-4  s-1 ) and Δx≠ (=8.3±0.1 Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.

Spike-In Normalization of ChIP Data Using DNA-DIG-Antibody Complex.

Chromatin immunoprecipitation (ChIP) is a widely used method to determine the occupancy of specific proteins within the genome, helping to unravel the function and activity of specific genomic regions. In ChIP experiments, normalization of the obtained data by a suitable internal reference is crucial. However, particularly when comparing differently treated samples, such a reference is difficult to identify. Here, a simple method to improve the accuracy and reliability of ChIP experiments by the help of an external reference is described. An artificial molecule, composed of a well-defined digoxigenin (DIG) labeled DNA fragment in complex with an anti-DIG antibody, is synthesized and added to each chromatin sample before immunoprecipitation. During the ChIP procedure, the DNA-DIG-antibody complex undergoes the same treatments as the chromatin and is therefore purified and quantified together with the chromatin of interest. This external reference compensates for variability during the ChIP routine and improves the similarity between replicates, thereby emphasizing the biological differences between samples.

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.

Nonradioactive Detection of Small RNAs Using Digoxigenin-Labeled Probes.

Small RNAs have been traditionally detected and quantified using small RNA blots, a modified Northern blot technique. The small RNAs are size-fractionated from the rest of the cellular RNA molecules by polyacrylamide gel electrophoresis and transferred by blotting onto a positively charged membrane. A radiolabeled probe was then traditionally used to detect a specific small RNA in the cellular pool. Small RNA blotting is a relatively simple, inexpensive approach to visualize small RNAs without artifacts. However, the radioactive labeling of the probe is sometimes an impediment, especially due to the requirement of specialized facilities. Here we describe a sensitive and simple method to detect and quantify small RNAs using digoxigenin-based nonradioactive RNA blots.

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.

Aptabody-aptatope interactions in aptablotting assays.

We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein-aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody-aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.

SDR-ELISA: Ultrasensitive and high-throughput nucleic acid detection based on antibody-like DNA nanostructure.

An ultrasensitive and high-throughput nucleic acid detection system, termed as strand displacement reaction-enzyme linked immunosorbent assay (SDR-ELISA), has been developed on the basis of antibody-like DNA nanostructures. Three digoxigenin or biotin modified hairpin probes are utilized to construct antibody-like DNA nanostructures that feature affinity toward streptavidin and anti-digoxigenin antibody via isothermal target-triggered SDR amplification. These antibody-like nanostructures have been employed to conjugate horseradish-peroxidase-labeled anti-digoxigenin antibody with streptavidin that is immobilized on microliter plate wells for enzyme-linked colorimetric assay. The resulting SDR-ELISA system is ultrasensitive for target DNA with a low detection limit of 5 fM. Moreover, the SDR-ELISA system is capable of discriminating DNA sequences with single base mutations, and do so in a high-throughput manner by detection and quantification of up to 96 or 384 DNA samples in a single shot. This detection system is further applied to detect other DNA targets such as Shewanella oneidensis specific DNA sequence, which indicates the generality of proposed SDR-ELISA system. The integration of SDR amplification and convenient ELISA technique advances an intelligent strategy for ultrasensitive and high-throughput nucleic acid detection, which may be amenable for direct visual detection and quantification using an accompanying quantitative color chart.

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.