Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions.

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

Development of a Simple Assay Method for Adenosine Deaminase via Enzymatic Formation of an Inosine-Tb3+ Complex.

Adenosine deaminase (ADA), which catalyzes the irreversible deamination of adenosine to inosine, is related to various human diseases such as tuberculous peritonitis and leukemia. Therefore, the method used to detect ADA activity and screen the effectiveness of various inhibitor candidates has important implications for the diagnosis treatment for various human diseases. A simple and rapid assay method for ADA, based on the enzymatic formation of a luminescent lanthanide complex, is proposed in this study. Inosine, an enzymatic product of ADA with stronger sensitization efficiency for Tb3+ than adenosine, produced a strong luminescence by forming an inosine-Tb3+ complex, and it enabled the direct monitoring of ADA activity in real-time. By introducing only Tb3+ to adenosine and ADA in the buffer, the enhancement of luminescence enabled the detection of a low concentration of ADA (detection limit 1.6 U/L). Moreover, this method could accurately determine the inhibition efficiency (IC50) of the known ADA inhibitor, erhythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and the inhibition of ADA could be confirmed by the naked eye. Considering its simplicity, this assay could be extended to the high-throughput screening of various ADA inhibitor candidates.

DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

A label-free kissing complexes-induced fluorescence aptasensor using DNA-templated silver nanoclusters as a signal transducer.

Riboswitches are complex folded RNA domains that serve as receptors for specific metabolites which identified in prokaryotes. They are comprised of a biosensor that includes the binding site for a small ligand and they respond to association with this ligand by undergoing a conformational change. In the present study, we report on the integration of silver nanoclusters (AgNCs) and riboswitches for the development of a kissing complexes-induced aptasensor (KCIA). We specifically apply the tunable riboswitches properties of this strategy to demonstrate the multiplexes analysis of adenosine and adenosine deaminase (ADA). This strategy allows for simple tethering of the specific oligonucleotides stabilizing the AgNCs to the nucleic acid probes. This is a new concept for aptasensors, and opens an opportunity for design of more novel biosensors based on the kissing complexes-induced strategy.

Adenosine deaminase biosensor combining cationic conjugated polymer-based FRET with deoxyguanosine-based photoinduced electron transfer.

We demonstrated a sensitive and selective adenosine deaminase (ADA) detection by modulating the fluorescence resonance energy transfer (FRET) between cationic conjugated poly(9,9-bis(6'-N,N,N-trimethylammonium) hexyl)fluorine phenylene) (PFP) and the deoxyguanosine-tailored hairpin aptamer. The hairpin aptamer was labeled with a fluorophore FAM at one end and three deoxyguanosines (Gs) at the other end as a quencher. In the absence of ADA, aptamer forms hairpin-like conformation with adenosines making close affinity of Gs and FAM, which results in the weak FRET from PFP to FAM because of FAM fluorescence being quenched by Gs via photoinduced electron transfer (PET). After addition of ADA, adenosine was hydrolyzed by ADA, followed by the release of free aptamer. In this case, FAM being far away from Gs, the strong FRET thus was obtained due to the quenching process being blocked. Therefore, the new strategy based on the FRET ratio enhancement is reasonably used to detect the ADA sensitively, combining the fluorescence signal amplification of conjugated polymers with the initiative signal decreasing by Gs. The detection limit of the ADA assay is 0.3 U/L in both buffer solution and human serum, which is more sensitive than most of those previously documented methods. Importantly, the assay is rapid, homogeneous, and simple without a complicated treating process. The ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), was also studied based on this assay, and the detection limit of EHNA is 10 pM. This strategy provides a new platform for the detection of other biomolecules and enzymes.

DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.

A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies.

Expression of a functional human adenosine deaminase in transgenic tobacco plants.

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5' UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.

An enzyme substrate binding aptamer complex based time-resolved fluorescence sensor for the adenosine deaminase detection.

In this work, we report an enzyme substrate binding aptamer complex based fluorescence sensor for an enzyme activity detection of adenosine deaminase (ADA). The sensor employs a DNA probe containing an adenosine aptamer region dually labeled with biotin and digoxigenin (DIG). The probe is immobilized in a streptavidin-modified 96-well micro plate via biotin-avidin bridge, and the DIG serves as an affinity tag for an Anti-DIG antibody conjugated with horseradish peroxidase (anti-DIG-HRP). The principle of our sensor is as follows: the aptamer forms a coiled structure making the DNA in a "closed" state in the presence of adenosine, which shields the DIG tag from the bulky anti-DIG-HRP due to a proper steric effect. After adding ADA in the test solution, adenosine will be converted to inosine regardless of the aptamer binding. Then, the inosine release causes the DNA to relax and consequently, the DIG becomes accessible to the bulky anti-DIG-HRP which will further conjugate a Eu³âº labeled anti-horseradish peroxidase (Eu-anti-HRP). The Eu-anti-HRP can give a fluorescence signal when an enhancement solution is added. In the result of the experiment, we found the sensor signal can reflect the enzyme activity accurately and the detection limit is lowered to 0.5 U L⁻¹ of ADA not only in buffer solution, but also in serum, and an enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride is studied. With a concentration of 0.01 nM it is enough to cause a distinct difference of the sensor response.

Purification and characterization of Plasmodium yoelii adenosine deaminase.

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 µM and 34 µM for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 µM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-ß-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/ß barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.

Complex of dipeptidyl peptidase II with adenosine deaminase.

Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).

Fasciola gigantica: purification and characterization of adenosine deaminase.

Nucleotidase cascades (apyrase, 5' nucleotidase, and adenosine deaminase (ADA) were investigated in the parasitic trematode Fasciola gigantica. ADA had the highest activity in the nucleotidase cascades. Adenosine deaminase was purified from F. gigantica through acetone precipitation and chromatography on CM-cellulose. Two forms of enzyme (ADAI, ADAII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass was 29 KDa for the native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme (ADAII) had a pH optimum at 7.5 and a K(m) 1.0 mM adenosine, a temperature optimum at 40 degrees C and heat stability up to 40 degrees C. The order of effectiveness of metals as inhibitors was found to be Hg(2+)>Mn(2+)>Cu(2+)>Ca(2+)>Zn(2+)>Ni(2+)>Ba(2+).

Transition state structure of E. coli tRNA-specific adenosine deaminase.

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.

Purification and assay of ADAR activity.

ADAR editing enzymes are found in all multicellular animals and are conserved in sequence and protein organization. The number of ADAR genes differs between animals, ranging from three in mammals to one in Drosophila. ADAR is also alternatively spliced to generate isoforms that can differ significantly in enzymatic activity. Therefore, to study the enzyme in vitro, it is essential to have an easy and reliable method of expressing and purifying recombinant ADAR protein. To add to the complexity of RNA editing, the number of transcripts that are edited by ADARs differs in different organisms. In humans there is extensive editing of Alu sequences, whereas in invertebrates transcripts expressed in the central nervous system are edited and this editing increases during development. It is possible to quantify site-specific RNA editing by sequencing of clones derived from RT-PCR products. However, for routine assaying of an edited position within a particular transcript, this is both expensive and time consuming. Therefore, a nonradioactive method based on poison primer extension assay is an ideal alternative.

Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies.

Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.

Adenosine ecto-deaminase (ecto-ADA) from porcine cerebral cortex synaptic membrane.

We have purified and investigated the role of adenosine ecto-deaminase (ecto-ADA) in porcine brain synaptic membranes and found a low activity of ecto-ADA in synaptic preparations from the cerebral cortex, hippocampus, striatum and medulla oblongata in the presence of purine transport inhibitors (NBTI, dipyridamole and papaverine). The purification procedure with affinity chromatography on epoxy-Toyopearl gel/purine riboside column as a crucial step of purification allowed a 214-fold purification of synaptic ecto-ADA with a yield of 30%. Gel filtration chromatography revealed a molecular mass estimated at 42.4+/-3.9 kDa. The enzyme had a broad optimum pH and was not affected by mono- and divalent cations. Ecto-ADA revealed a low affinity to adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) (K(M)=286.30+/-40.38 microM and 287.14+/-46.50 microM, respectively). We compared the affinity of ecto-ADA to the substrates with the physiological and pathological concentrations of the extracellular Ado in brains that do not exceed a low micromolar range even during ischemia and hypoxia, and with the affinity of adenosine receptors to Ado not exceeding a low nanomolar (A(1) and A(2A) receptors) or low micromolar (A(2B) and A(3)) range. Taken together, our data suggest that the role of synaptic ecto-ADA in the regulation of the ecto-Ado level in the brain and in the termination of adenosine receptor signaling is questionable. The porcine brain synapses must have other mechanisms for the ecto-Ado removal from the synaptic cleft and synaptic ecto-ADA may also play an extra-enzymatic role in cell adhesion and non-enzymatic regulation of adenosine receptor activity.

Adenosine deaminase from camel tick Hyalomma dromedarii: purification and characterization.

Adenosine deaminase is involved in purine metabolism and is a key enzyme for the control of the cellular levels of adenosine. Adenosine deaminase activity showed significant changes during embryogenesis of the camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-sepharose, three forms of enzyme (ADAI, ADAII and ADAIII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass of adenosine deaminase ADAII was 42 kDa for the native enzyme and represented a monomer of 42 kDa by SDS-PAGE. The enzyme had a pH optimum at 7.5 and temperature optimum at 40 degrees C with heat stability up to 40 degrees C. ADAII had a K (m) of 0.5 mM adenosine with higher affinity toward deoxyadenosine and adenosine than other purines. Ni(2+), Ba(2+), Zn(2+), Li(2+), Hg(2+) and Mg(2+) partially inhibited the ADAII. Mg(2+) was the strongest inhibitor by 91% of the enzyme's activity.

Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase.

The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-)(1) and K(M) = 0.83 +/- 0.22 microM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 microM). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

Human plasma adenosine deaminase 2 is secreted by activated monocytes.

Adenosine deaminase (ADA) plays an important role in the immune system, and its activity is composed of two kinetically distinct isozymes, ADA1 and ADA2. ADA2 is a major component of human plasma total ADA activity and ADA2 activity is significantly elevated in patients with various diseases such as HIV infection and chronic hepatitis. However, relatively little is known about ADA2 because of difficulties in purifying this enzyme. In this study we succeeded in purifying human plasma ADA2 and demonstrate the extracellular secretion of ADA2 from human peripheral blood monocytes stimulated with phorbol 12-myristate 13-acetate and calcium ionophore.

A method to find tissue-specific novel sites of selective adenosine deamination.

Site-selective adenosine (A) to inosine (I) RNA editing by the ADAR enzymes has been found in a variety of metazoan from fly to human. Here we describe a method to detect novel site-selective A to I editing that can be used on various tissues as well as species. We have shown previously that there is a preference for ADAR2-binding to selectively edited sites over non-specific interactions with random sequences of double-stranded RNA. The method utilizes immunoprecipitation (IP) of intrinsic RNA-protein complexes to extract substrates subjected to site-selective editing in vivo, in combination with microarray analyses of the captured RNAs. We show that known single sites of A to I editing can be detected after IP using an antibody against the ADAR2 protein. The RNA substrates were verified by RT-PCR, RNase protection and microarray. Using this method it is possible to uniquely identify novel single sites of selective A to I editing.