RNA Heteroduplex Research Articles

Subcellular location of L1 retrotransposon-encoded ORF1p, reverse transcription products, and DNA sensors in lupus granulocytes

BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease with an unpredictable course of recurrent exacerbations alternating with more stable disease. SLE is characterized by broad immune activation and autoantibodies against double-stranded DNA and numerous proteins that exist in cells as aggregates with nucleic acids, such as Ro60, MOV10, and the L1 retrotransposon-encoded ORF1p.ResultsHere we report that these 3 proteins are co-expressed and co-localized in a subset of SLE granulocytes and are concentrated in cytosolic dots that also contain DNA: RNA heteroduplexes and the DNA sensor ZBP1, but not cGAS. The DNA: RNA heteroduplexes vanished from the neutrophils when they were treated with a selective inhibitor of the L1 reverse transcriptase. We also report that ORF1p granules escape neutrophils during the extrusion of neutrophil extracellular traps (NETs) and, to a lesser degree, from neutrophils dying by pyroptosis, but not apoptosis.ConclusionsThese results bring new insights into the composition of ORF1p granules in SLE neutrophils and may explain, in part, why proteins in these granules become targeted by autoantibodies in this disease.

Novel Insights into MEG3/miR664a-3p/ADH4 Axis and Its Possible Role in Hepatocellular Carcinoma from an in Silico Perspective

Hepatocellular carcinoma (HCC) is a complex disease involving altered interactomes of transcripts and proteins. MicroRNAs (miRNAs) are small-noncoding RNAs that can interact with specific gene transcripts and an array of other vital endogenous non-coding RNAs (lncRNAs) that can influence gene expression. Maternally Expressed Gene 3 (MEG3) is an imprinted lncRNA that is reported to be downregulated in HCC (in both cell lines and tumors). Alcohol Dehydrogenase 4 (ADH4) is a well-known prognostic protein biomarker for predicting the survival outcomes of patients with hepatocellular carcinoma whose expression is regulated by miR-664a-3p, which is upregulated in HCC. In this study, we performed a battery of robust and systematic in silico analyses to predicate the possible lncRNA-miRNA interactions between MEG3, miR-664a-3p, and ADH4. miRNA-mRNA and lncRNA-miRNA hybrid structures were primarily obtained, and the minimum free energies (MFEs) for the 3'UTR (Untranslated Regions) of ADH4-miR-664a-3p and the 3'UTR of MEG3-miR-664a-3p interactions were assessed to predict the stability of the obtained RNA heteroduplex hybrids. The hybrid with the least minimum free energy (MFE) was considered to be the most favorable. The MFEs were around -28.1 kcal/mol and -31.3 kCal/mol for the ADH4-miR-664a-3p and MEG3-miR-66a-3p RNA hybrids, respectively. This demonstrated that lncRNA-MEG3 might be a competitive endogenous RNA that acts as a molecular sponge for miR-664a-3p. In summary, our interaction analyses results predict the significance of the MEG3/miR-664a-3p/ADH4 axis, where MEG3 downregulation results in miR-664a-3p overexpression and the subsequential underexpression of ADH4 in HCC, as a novel axis of interest that demands further validation.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Systemic CLIP-seq analysis and game theory approach to model microRNA mode of binding.

microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.

Advances in the Detection of Toxic Algae Using Electrochemical Biosensors.

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L−1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification

DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

The 40S small ribosomal subunit is cotranscriptionally assembled in the nucleolus as part of a large chaperone complex called the 90S preribosome or small-subunit processome. Here, we present the 3.2-Å-resolution structure of the Chaetomium thermophilum 90S preribosome, which allowed us to build atomic structures for 34 assembly factors, including the Mpp10 complex, Bms1, Utp14 and Utp18, and the complete U3 small nucleolar ribonucleoprotein. Moreover, we visualized the U3 RNA heteroduplexes with a 5' external transcribed spacer (5' ETS) and pre-18S RNA, and their stabilization by 90S factors. Overall, the structure explains how a highly intertwined network of assembly factors and pre-rRNA guide the sequential, independent folding of the individual pre-40S domains while the RNA regions forming the 40S active sites are kept immature. Finally, by identifying the unprocessed A1 cleavage site and the nearby Utp24 endonuclease, we suggest a proofreading model for regulated 5'-ETS separation and 90S-pre-40S transition.

Electrochemical immunosensor based on hairpin DNA probe for specific detection of N6-methyladenosine RNA

N6-methyladenosine (m6A) has been identified as the most frequent messenger RNA (mRNA) modification which is the reversible process of RNA methylation modification and related to biological rhythm regulation. In this work, an electrochemical immunosensor was developed for RNA methylation detection via using gold nanoparticles modified Au electrode (AuNPs/Au) as substrate electrode. Firstly, the hairpin DNA probe was assembled on the surface of AuNPs/Au via AuS bond. In the presence of the hairpin DNA, which is the template of assistant DNA and target RNA, the ligase can selectively ligate them as the template of the hairpin DNA to form a long DNA: RNA heteroduplex strand. Successively, anti-N6-methyladenosine antibody (anti-m6A antibody) could be specifically conjugated onto the RNA methylation site, and the horseradish peroxidase labeled goat anti mouse IgG (HRP-IgG) was further conjugated onto the anti-m6A antibody. In the detection buffer solution with H2O2 and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation to generate benzoquinone, resulting in a highly electrochemical reduction signal. This developed immunosensor shows a wide linear range for methylated RNA from 10fM to 10nM, low detection limit of 3.35fM (S/N=3), high specificity and satisfactory reproducibility. And this immunosensing method owns great potentials for providing the assay of the expression level of methylated RNA in micro-biological system.

Simultaneous detection of two hepatocellar carcinoma-related microRNAs using a clever single-labeled fluorescent probe

The simultaneous detection of two important microRNAs (miRNAs) can potentially evaluate pathological states. The simultaneous detection of miRNA-26a and miRNA-122 is proposed in this study using a cleverly designed single-labeled, dual-functional fluorescent probe with a 2-aminopurine as a fluorophore, which is a G-quadruplex single-stranded DNA. The probe can partly complement cDNA-1 and cDNA-2. cDNA-1 and cDNA-2 are complementary strands of miRNA-26a and miRNA-122, respectively. When the target miRNAs are added simultaneously, these cDNA (cDNA-1 and cDNA-2) can be competed off from the cDNA∖probe duplex to form a cDNA∖RNA heteroduplex. The probe is released and the fluorescent signal is increased. The detection limits of miRNA-26a and miRNA-122 are both 2.5 nM, and their wide linear which ranges from 2.5 to 500 nM are achieved using the assay. This method has potential practical applications.

VirF Relieves the Transcriptional Attenuation of the Virulence Gene icsA of Shigella flexneri Affecting the icsA mRNA-RnaG Complex Formation.

VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF–RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to “fish” out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5′-UUUUaGYcUuUau-3′) that is the RNA-converted consensus sequence previously proposed for the VirF–DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF–RNA interaction, in particular when exposed in a stem–loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA–RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem–loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA–RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.

How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg2+ ion availability at the cleavage site causing sequence-specific hydrolysis of the phosphodiester bond of scissile phosphate. The enhancement of the total rate of cleavage of the complementary mRNA strand by up to 25%, presented in this work, provides opportunities to engineer a single modification site in appropriately substituted AONs to design an effective antisense strategy based on the nucleolytic stability of the AON strand versus RNase H capability to cleave the complementary RNA strand.

Double-Strand Displacement Biosensor and Quencher-Free Fluorescence Strategy for Rapid Detection of MicroRNA.

We describe a facile quencher-free fluorescence strategy for rapid detection of microRNAs (miRNAs) by using a novel double-strand displacement sensor. The sensor is designed with an outstanding 2-aminopurine (2-AP) fluorophore as a probe and a predesigned cDNA, which can completely complement the target miRNA and partly complement the 2-AP probe. When the target miRNA is added, the cDNA can be competed off from the cDNA\2-AP probe duplex, thereby forming a cDNA\RNA heteroduplex. The free 2-AP probe induces an increase in the fluorescent signal. A limit of detection of 5 nM and a wide linear range from 5 to 1000 nM (R(2) = 0.9971) are achieved by this assay. The rapid detection strategy can be accomplished within 2 h without expensive nanoparticles and complicated instruments for the whole procedure, thus, offering a significant potential for clinical application.

Abstract B30: Rnaseh2c is a candidate metastasis susceptibility gene in breast cancer

Abstract Breast cancer is a leading cause of cancer-related female deaths in the United States, the majority of which are due to distant metastases. Elucidating novel genes that drive metastasis is critical to improving patient outcome. Our laboratory has extensively shown that germline variants impact an individual's susceptibility for metastasis, and we have used this to identify novel metastasis modifier genes. Here we applied a mouse model of spontaneous metastasis to a mouse breeding scheme to screen for strain-specific haplotypes associated with metastatic phenotype. Additional filtering of the haplotype-containing genes was performed for those that were highly expressed in tumors. This screen identified Rnaseh2c, a scaffolding subunit of the Ribonuclease H2 complex which degrades RNA from DNA:RNA heteroduplexes such as those generated during replication and transcription, thus helping maintain genomic stability. Though mutations in RNASEH2C are known to cause the neurological autoinflammatory disorder Aicardi-Goutières Syndrome (AGS), it has not previously been linked to cancer. Given the known pathology of AGS, we hypothesize that differential expression of Rnaseh2c in tumor cells alters tumor progression and metastasis potentially via interaction with the immune system. We directly assessed the effect of tumor cell Rnaseh2c expression on metastasis using in vivo metastasis assays. We implanted into the mammary fat pad tumor cells expressing shRNAs that reduced Rnaseh2c expression and observed significantly fewer pulmonary metastases and reduced tumor burden. To complement this approach, we performed the same metastasis assay with cells overexpressing exogenous Rnaseh2c. In agreement with our initial result, overexpression generated significantly more metastases without affecting primary tumor size, confirming Rnaseh2c as a metastasis modifier. The results thus far validate our approach of employing germline variation in mice to identify novel metastasis modifier genes. Current and future work will focus on elucidating the mechanism by which Rnaseh2c expression impacts metastasis. Specifically, we will determine where Rnaseh2c functions in the metastatic cascade, whether modulated expression disrupts Rnase H2 complex formation, and whether the immune system is involved in mediating this gene's effect on metastasis. Elucidating the mechanism by which this gene acts can contribute to both the development of novel metastasis biomarkers as well as potential therapeutic targets. Citation Format: Sarah Deasy, Kent Hunter. Rnaseh2c is a candidate metastasis susceptibility gene in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B30.

The first crystal structures of RNA-PNA duplexes and a PNA-PNA duplex containing mismatches--toward anti-sense therapy against TREDs.

PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA–PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA–PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA–RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson–Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA.

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair.

A dual-amplified electrochemical detection of mRNA based on duplex-specific nuclease and bio-bar-code conjugates

On the basis of strong preference for cleaving double-stranded DNA or DNA in DNA:RNA heteroduplexes of duplex-specific nuclease (DSN), a dual-amplified electrochemical detection of mRNA was developed in this article, by coupling the enhancement of DSN and bio-bar-code conjugates. Capture probe was linked with magnetic nanoparticles (MNPs) at its 5′ end and bio-bar-code at its 3′ end. In the presence of target surviving mRNA, all hybridized S1 strands were cleaved off the biosensor by the DSN, and the bio-bar-code probe with CdS nanoparticles (CdS NPs) was released into the solution. The metal sulfide nanoparticles were measured by anodic stripping voltammetry (ASV) subsequently. This assay exhibited high sensitivity and selectivity with a detection limit of 0.48fM. In addition, we proved that this simple and cost-effective strategy is capable of detecting the target in complicated biological samples and holds great potential application in biomedical research and clinical diagnostics.

Mechanism and Manipulation of DNA:RNA Hybrid G-Quadruplex Formation in Transcription of G-Rich DNA

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.

Highly selective detection of microRNA based on distance-dependent electrochemiluminescence resonance energy transfer between CdTe nanocrystals and Au nanoclusters

A distance-dependent electrochemiluminescence resonance energy transfer (ERET) system based on CdTe nanocrystals and Au nanoclusters (Au NCs) was designed with the aid of ligase for highly selective detection of microRNA (miRNA). First, Au NCs functionalized hairpin DNA was synthesized via Au–S chemistry, and characterized with transmission electron microscopy and dynamic light scattering. The resulting hairpin DNA–Au NCs composite can be bound to the carboxylated CdTe nanocrystals via amide reaction on glass carbon electrode. The strong interaction between CdTe nanocrystals and AuNCs led to the electrochemiluminescence (ECL) quenching of CdTe nanocrystals. In the presence of assistant DNA and miRNA, the ligase can selectively ligate both of them on the strand of the hairpin DNA to form long DNA–RNA heteroduplexes. Thus the ECL signal was recovered due to the blocking of the ERET. As a comparison, when directly opening the hairpin DNA by the target, the ECL emission signal is weak owing to the presence of ERET effect at the short distance. Based on the distance-dependent ERET, a ‘signal on’ ECL system was utilized for the detection of miRNA with the advantages of 6 orders magnitude linear range and excellent sequence specificity. The total detection processing time of the biosensor was approximately 70min. By substituting the hairpin DNA with different sequences, this strategy as a new signal transduction approach could be conveniently extended for detection of other short miRNA and DNA.

New Anthraquinone Derivatives as Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Function

Background: The degradative activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), termed ribonuclease H (RNase H), which hydrolyzes the RNA component of the heteroduplex RNA:DNA replication intermediate, is an excellent target for drug discovery. Anthraquinones (AQs) and their derivatives, which are common secondary metabolites occurring in bacteria, fungi, lichens and a large number of families in higher plants, have been reported to have several biological activities including that of inhibiting HIV-1 RT activities in biochemical assays. Methods: We have assayed new AQ derivatives on HIV-1 RNase H activities in biochemical assays. Results: Six series of new AQ derivatives with various substituents at positions 1, 2, 3 and 4 of the AQ ring were tested, and new analogs able to inhibit HIV-1 RT-associated RNase H activity in the low micromolar range were found. Conclusions: Our results demonstrate that AQ derivatives are promising anti-RNase H inhibitors.