CRISPR/Cas12a collateral cleavage activity for an ultrasensitive assay of RNase H

We herein describe an ultrasensitive RNase H assay by utilizing CRISPR/Cas12a collateral cleavage activity. In this technique, a chimeric duplex probe consisting of activator DNA and its complementary blocker RNA...

Communication and Cross-Regulation Between Multiple Concatenated Enzymatic Reaction Networks

Nature connects multiple fuel-driven chemical/enzymatic reaction networks (CRNs/ERNs) via cross-regulation to hierarchically control biofunctions for a tailored adaption in complex sensory landscapes. In contrast, emerging artificial fuel-driven systems most-ly focus on a single CRN and their implementation to direct self-assembly or material responses. In this work, we introduce a facile example of communication and cross-regulation among multiple DNA-based ERNs regulated by a concatenated RNA transcription regulator. For this purpose, we run two fuel-driven DNA-based ERNs by concurrent NAD+-fueled ligation and restriction via endo-nucleases (REases) in parallel. ERN one allows for the dynamic steady-state formation of the promoter sequence for T7 RNA poly-merase, which activates RNA transcription. The produced RNA regulator can repress or promote the second ERN via RNA-mediated strand displacement. Furthermore, adding RNase H to degrade the produced RNA can restart the reaction or tune the lag time of two ERNs, giving rise to a repression-recovery and promotion-stop processes. We believe that concatenation of multiple CRNs provides a basis for the design of more elaborate autonomous regulatory mechanisms in systems chemistry and synthetic biology.

Evaluation of Different Mycobacterial Species for Drug Discovery and Characterisation of Novel Inhibitors of Mycobacterium Tuberculosis

<p>Tuberculosis (Tb) has plagued mankind for many centuries and is still a leading cause of death worldwide. A worrying development is the emergence of drug-resistant Tb that poses further challenges to the control of the disease. The global Tb burden and high mortality rate indicate that new drugs are needed for Tb treatment. While no new anti-Tb agents have been introduced to the market for about three decades, drugs with novel mechanisms of action can amend the current Tb treatment regimen and may provide an effective solution to drug resistance. The main objectives of this study were to identify an appropriate in vitro model that could be used for anti-Tb drug high-throughput screening (HTS), and to use this model to identify a novel candidate anti-tubercular drug and its cognate cellular target. A sensitive growth inhibition assay was set up with a GFP-labelled Tb vaccine strain, M. bovis BCG, using standard first and second line anti-tubercular drugs. HTS of the drug libraries was performed with various in vitro models to evaluate their efficacy for use in anti-Tb drug discovery. Approximately 50% of the M. tuberculosis inhibitors were not detected in screening with the surrogate species, M. smegmatis; whereas, only 21% of hits were not detected with M. bovis BCG. A comparative genomic study revealed that 97% of M. bovis BCG proteins, compared to 70% in M. smegmatis have conserved orthologues in M. tuberculosis H37Rv. Therefore, M. bovis BCG represented a more sensitive model than M. smegmatis for detecting anti-M. tuberculosis compounds. M. bovis BCG was then used to screen for novel anti-Tb agents by HTS of compound libraries and various plant extracts, followed by validation of new compounds in M. tuberculosis H37Ra. A number of novel M. tuberculosis inhibitors were identified, including sappanone A dimethyl ether from plant sources and compounds NSC112200 and NSC402959 from NIH chemical libraries. The inhibitors that were validated using M. tuberculosis H37Ra were also validated in the virulent Tb strain, M. tuberculosis H37Rv. In addition, their activity was further investigated using a suite of other clinically important human pathogens. One of the key anti-mycobacterial hits identified in this study, NSC402959, has previously been detected in screens for compounds that inhibit ribonuclease H (RNase H), an enzyme that is required for a number of essential cellular processes. NSC402959 inhibited RNase H proteins from HIV as well as from E. coli. Since HIV and Tb are major pandemics, previously-known anti-HIV RNase H compounds were imported and tested for their anti-proliferative activity towards M. tuberculosis H37Ra. HIV RNase H inhibitors, NSC35676, NSC112200, NSC133457 and NSC668394, exhibited good anti-mycobacterial activity in this study. In silico analysis suggested a plausible interaction of these inhibitors with mycobacterial RNase HI. A biochemical assay further confirmed NSC112200 to be specific against RNase HI from M. tuberculosis. These interesting inhibitors were not only structurally different from existing anti-Tb drugs but some of them were also non-toxic to mammalian cells and may have a unique mechanism of action. Thus, these compounds showed good potential for development as dual inhibitors of Tb and HIV; therefore, future studies in animal infection models to determine their dual anti-mycobacterial and anti-HIV activities are warranted.</p>

Evaluation of Different Mycobacterial Species for Drug Discovery and Characterisation of Novel Inhibitors of Mycobacterium Tuberculosis

<p>Tuberculosis (Tb) has plagued mankind for many centuries and is still a leading cause of death worldwide. A worrying development is the emergence of drug-resistant Tb that poses further challenges to the control of the disease. The global Tb burden and high mortality rate indicate that new drugs are needed for Tb treatment. While no new anti-Tb agents have been introduced to the market for about three decades, drugs with novel mechanisms of action can amend the current Tb treatment regimen and may provide an effective solution to drug resistance. The main objectives of this study were to identify an appropriate in vitro model that could be used for anti-Tb drug high-throughput screening (HTS), and to use this model to identify a novel candidate anti-tubercular drug and its cognate cellular target. A sensitive growth inhibition assay was set up with a GFP-labelled Tb vaccine strain, M. bovis BCG, using standard first and second line anti-tubercular drugs. HTS of the drug libraries was performed with various in vitro models to evaluate their efficacy for use in anti-Tb drug discovery. Approximately 50% of the M. tuberculosis inhibitors were not detected in screening with the surrogate species, M. smegmatis; whereas, only 21% of hits were not detected with M. bovis BCG. A comparative genomic study revealed that 97% of M. bovis BCG proteins, compared to 70% in M. smegmatis have conserved orthologues in M. tuberculosis H37Rv. Therefore, M. bovis BCG represented a more sensitive model than M. smegmatis for detecting anti-M. tuberculosis compounds. M. bovis BCG was then used to screen for novel anti-Tb agents by HTS of compound libraries and various plant extracts, followed by validation of new compounds in M. tuberculosis H37Ra. A number of novel M. tuberculosis inhibitors were identified, including sappanone A dimethyl ether from plant sources and compounds NSC112200 and NSC402959 from NIH chemical libraries. The inhibitors that were validated using M. tuberculosis H37Ra were also validated in the virulent Tb strain, M. tuberculosis H37Rv. In addition, their activity was further investigated using a suite of other clinically important human pathogens. One of the key anti-mycobacterial hits identified in this study, NSC402959, has previously been detected in screens for compounds that inhibit ribonuclease H (RNase H), an enzyme that is required for a number of essential cellular processes. NSC402959 inhibited RNase H proteins from HIV as well as from E. coli. Since HIV and Tb are major pandemics, previously-known anti-HIV RNase H compounds were imported and tested for their anti-proliferative activity towards M. tuberculosis H37Ra. HIV RNase H inhibitors, NSC35676, NSC112200, NSC133457 and NSC668394, exhibited good anti-mycobacterial activity in this study. In silico analysis suggested a plausible interaction of these inhibitors with mycobacterial RNase HI. A biochemical assay further confirmed NSC112200 to be specific against RNase HI from M. tuberculosis. These interesting inhibitors were not only structurally different from existing anti-Tb drugs but some of them were also non-toxic to mammalian cells and may have a unique mechanism of action. Thus, these compounds showed good potential for development as dual inhibitors of Tb and HIV; therefore, future studies in animal infection models to determine their dual anti-mycobacterial and anti-HIV activities are warranted.</p>

The human TRAM1 locus expresses circular RNAs

Numerous indirect and in silico produced evidences suggest circular RNAs (circRNA) in mammals while thorough experimental proofs of their existence have rarely been reported. Biological studies of circRNA, however, should be based on experimentally verified circRNAs. Here, we describe the identification of two circRNAs originating from the gene locus of the translocation associated membrane protein 1 (TRAM1). Linear and potentially circular TRAM1-specific transcripts were identified in a transcriptome analysis of urine RNA of bladder cancer (BCa) patients versus healthy donors. Thus, we first focused on the topology of TRAM1-specific transcripts. We describe conclusive experimental evidence for the existence of TRAM1-specific circRNAs in the human BCa cell lines ECV-304 and RT-4. PCR-based methodology followed by cloning and sequencing strongly indicated the circular topology of two TRAM1 RNAs. Further, studies with exon fusion sequence-specific antisense oligonucleotides (asON) and RNase H as well as studies in the use of RNase R contribute to conclusive set of experiments supporting the circular topology of TRAM1 transcripts. On the biological side, TRAM1-specific circRNAs showed low expression levels and minor differences in BCa cell lines while linear TRAM1 transcripts displayed down-regulated expression in the higher cancer stage model ECV-304 versus more differentiated RT-4 cells.

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Prime editing is a new CRISPR-based genome editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with AAV viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4x fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. This novel minimized PE provides great value to AAV-based delivery applications in vivo.

Exploiting Transcription-Replication Conflicts As a Novel Therapeutic Intervention in Multiple Myeloma

Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained in malignant PCs meaning that these cells display an elevated transcriptional stress. Besides, malignant PCs face oncogene-induced replication stress concomitantly with cell cycle deregulation. Consequently, transcription and replication in malignant PCs need to be tightly coordinated to avoid too much interferences that would increase replication stress and genomic instability. A failure to cope with these transcription/replication conflicts (TRCs) could have a significant impact on mutagenesis involved in MM development. Importantly, these effects might open the therapeutic possibility of TRCs enhancement to specifically kill malignant PCs. Based on these observations, we identified a signature of 13 TRCs resolution factors significantly overexpressed in MM patients. Considering the potent role of TRCs resolution in MM cell adaptation to replication stress, we sought to identify the TRCs resolution factors that are associated with a poor outcome in MM. High expression of 9 out of the 13 TRCs resolution factors significantly overexpressed in malignant PCs are associated with a poor outcome in MM (TT2 cohort, n = 345). We gathered the prognostic value of these 9 genes within a Gene Expression Profile (GEP)-based TRC resolution score (TRC score). High TRC score is associated with a poor outcome in two independent cohorts of newly diagnosed MM patients treated by high dose therapy and autologous stem cell transplantation (Arkansas, TT2 cohort, n = 345; CoMMpass cohort, n = 674) (Fig.1A). Interestingly, we investigated the link between the TRC score and the MM cells drug response using our collection of human myeloma cell lines (HMCLs), and identified that HMCLs with high TRC score values are significantly more sensitive to Panobinostat histone deacetylase inhibitor, currently used in MM treatment at relapse (n = 11, p value &lt; 0.05). Histone acetylation has been shown to promote R-loop formation that constitutes obstacles to replication fork progression. Using primary MM cells from patients (n = 12) co-cultured with their bone marrow microenvironment, we found that a high TRC score value is associated with a higher toxicity of Panobinostat (p value &lt; 0.01). Therefore, the TRC score allows the identification of a MM patients subgroup with a poor outcome that could benefit from Panobinostat treatment. Interestingly, TRCs are promoted by R-loop formation and G-quadruplex (G4) stabilizers treatment. R-loops are formed by the reannealing of the nascent RNA with the template DNA (called an RNA:DNA hybrid). G4s are four-stranded secondary DNA structures, constituted of stacked guanine tetrads. Both structures are formed during transcription in G-rich DNA regions and can represent a barrier for replication fork progression if unscheduled. G4s can stabilize R-loops which have been shown to mediate DNA damage induced by G4 stabilizers. Interestingly, treatment with the G4 stabilizer Pyridostatin (PDS) was associated with significant toxicity on HMCLs (n = 15) (Fig.1B), and on primary MM cells of patients cocultured with their bone marrow microenvironment (n = 5, p value &lt; 0.05). Interestingly, the combination of PDS and Panobinostat has a synergistic effect in HMCLs. We also found a correlation between HMCLs TRC score and the response to two Bromodomain and Extra-Terminal motif (BET) proteins inhibitors, I-BET-762 and RVX-208. The synergistic effect of PDS combination with I-BET-762 was validated in vitro. BET proteins inhibition has been shown to increase R-loop formation and DNA damage. Furthermore, we used inducible RNase H expression in HMCLs to specifically degrade RNA:DNA hybrids. RNase H expression resulted in a significant reduction of DNA damage response after PDS treatment (Fig.1C). Our results underline that spontaneous replication stress and genomic instability are related to R-loop formation and TRCs in MM cells. Altogether, these results emphasize the therapeutic potential of TRCs targeting in MM using G4 stabilizers alone or in combination with current treatments. Figure 1 Figure 1. Disclosures Vincent: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Cartron: Roche, Celgene-BMS: Consultancy; Danofi, Gilead, Novartis, Jansen, Roche, Celgene-BMS, Abbvie, Takeda: Honoraria. Herbaux: Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Moreaux: Diag2Tec: Consultancy.

Effects of troponoids on mitochondrial function and cytotoxicity

The α-hydroxytropolones (αHT) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target the HBV ribonuclease H (RNase H) with sub-micromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by MTS assays that assesses mitochondrial function. Earlier studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial:nuclear genome ratio, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass did interfere with these processes. The troponoids had no impact on the mitochondrial DNA to nuclear DNA ratio after three days of compound exposure. Patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines leading to cytotoxicity, but are not be cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by co-administration with ROS antagonists to ameliorate increased ROS levels.