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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.

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 Tb 3+ than adenosine, produced a strong luminescence by forming an inosine-Tb 3+ complex, and it enabled the direct monitoring of ADA activity in real-time. By introducing only Tb 3+ 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 (IC 50 ) 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.

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.