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Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

RNA–DNA hybrid is a part of the R‐loop which is an important non‐standard nucleic acid structure. RNA–DNA hybrid/R‐loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA–DNA hybrids and map their genomic locations in fission yeast cells. The RNA–DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild‐type. The majority of RNA–DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S‐phase and about 20% of the RNA–DNA hybrids overlap with Rad52 loci. During S‐phase, more robust association of Rad52 with RNA–DNA hybrids was observed in the protein coding region than in M‐phase. These results suggest that persistent RNA–DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S‐phase, potentially through collision with DNA replication forks. [ABSTRACT FROM AUTHOR]

Divergent banana streak viruses (BSV) were characterized from banana plants exhibiting diverse symptoms in the Northeast region (NER) of India. Using rolling circle amplification (RCA), the complete genome sequences of seven episomal banana streak MY virus (BSMYV) isolates, including two novel variants, and two new banana streak IM virus (BSIMV) isolates were characterized. The novel BSMYV genetic variants were associated with conspicuous necrosis on newly emerged leaves, peduncle distortion, pseudostem internal necrosis, in addition to common streak symptoms. For complete genome nucleotide sequences, BSMYV-IN4 and IN5 shared 77–79% identity with other BSMYVs, while BSMYV-IN7 and IN8 exhibited identities of 77–97%. This study reports for the first time, the complete genomes of two banana streak IM virus (BSIMV-IN1 and -IN2) infecting triploid banana hybrids exhibiting leaf distortion, stunted rosette-like growth, and necrosis, sharing 87% sequence identity with reference BSIMV genome (GenBank accession no. HQ593112). Phylogenetic inference based on complete genomes revealed the distinct and congruent placement of BSMYV-IN4 and IN5 within the BSMYV cluster. Pairwise sequence comparisons of the conserved RT/RNase H nucleotide (nt) sequences revealed that the BSMYV-IN7 and IN4 isolates showed 85% and 97% identity to BSMYV (AY805074), respectively, which shared highest nt identity with BSMYV-IN6, IN9, and IN10, at 100%. The RT/RNase H nt sequences of BSIMV-IN1 and IN2 had 98% identity with the BSIMV (HQ593112), but were characterized as novel variants of BSIMV based on complete genomes. An analysis of relative synonymous codon usage (RSCU) pattern in the ORFIII polyprotein of BSMYV and BSIMV isolates revealed AGA and AGG (arginine) as the most frequently overrepresented codons (>1.5), evolutionary conserved in the genome of both species. A total of 14 recombination events were detected among the 36 BSV genomes, with recombination breakpoints mainly located in the ORFI, III, and IGR genomic regions. A novel phylogenetic cluster, comprised of BSMYV-IN4 and IN5 within the clade I was probably derived from heterologous recombination between parents resembling banana streak VN virus (BSVNV; AY750155) and banana streak GF virus (BSGFV; KJ013507) isolates. The present study conclusively reports the infection of genetically and symptomatically distinct variants of BSMYV and BSIMV infecting banana hybrids in NER India. [ABSTRACT FROM AUTHOR]

Background: Aberrant accumulation of R-loops leads to DNA damage, genome instability and even cell death. Therefore, the timely removal of harmful R-loops is essential for the maintenance of genome integrity. Nucleolar R-loops occupy up to 50% of cellular R-loops due to the frequent activation of Pol I transcription. However, the mechanisms involved in the nucleolar R-loop resolution remain elusive. The nucleolar acetyltransferase NAT10 harbors a putative RecD helicase domain (RHD), however, if NAT10 acts in the R-loop resolution is still unknown. Methods: NAT10 knockdown cell lines were constructed using CRISPR/Cas9 technology and short hairpin RNA targeting NAT10 mRNA, respectively. The level of R-loops was detected by immunofluorescent staining combined with RNase H treatment. The helicase activity of NAT10 or DDX21 was determined by in vitro helicase experiment. The interaction between NAT10 and DDX21 was verified by co-immunoprecipitation, immunofluorescent staining and GST pull-down experiments. Acetylation sites of DDX21 by NAT10 were analyzed by mass spectrometry. NAT10 knockdown-induced DNA damage was evaluated by immunofluorescent staining and Western blot detecting γH2AX. Results: Depletion of NAT10 led to the accumulation of nucleolar R-loops. NAT10 resolves R-loops through an RHD in vitro and in cells. However, Flag-NAT10 ∆RHD mutant still partially reduced R-loop levels in the NAT10-depleted cells, suggesting that NAT10 might resolve R-loops through additional pathways. Further, the acetyltransferase activity of NAT10 is required for the nucleolar R-loop resolution. NAT10 acetylates DDX21 at K236 and K573 to enhance the helicase activity of DDX21 to unwind nucleolar R-loops. The helicase activity of DDX21 significantly decreased by Flag-DDX21 2KR and increased by Flag-DDX21 2KQ in cells and in vitro. Consequently, NAT10 depletion-induced nucleolar R-loop accumulation led to DNA damage, which was rescued by co-expression of Flag-DDX21 2KQ and Flag-NAT10 G641E, demonstrating that NAT10 resolves nucleolar R-loops through bipartite pathways. Conclusion: We demonstrate that NAT10 is a novel R-loop resolvase and it resolves nucleolar R-loops depending on its helicase activity and acetylation of DDX21. The cooperation of NAT10 and DDX21 provides comprehensive insights into the nucleolar R-loop resolution for maintaining genome stability. [ABSTRACT FROM AUTHOR]

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours' post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products. Key points: • The versatile sequence optimization to improve the yield of the recombinant enzyme. • Directed specific mutations can promise to produce thermostable and soluble RT commercial enzymes. • Predicted hot mutations in the active site of the RNase H domain of recombinant RT without any effect on its structure. [ABSTRACT FROM AUTHOR]

Deuterium (2H) spin relaxation of 13CH2D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B0 dependence of the 2H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ωD) and J(2ωD). In this study, the linear relation between 2H relaxation rates at B0 fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate 13C-12C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R1, R1ρ, RQ, RAP) were measured for 57 well-resolved 13CH2D moieties in RNase H at 1H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B0 fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ωD) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τM, Sf2, τf) or extended model-free function (τM, Sf2, Ss2, τf, τs) to obtain optimized dynamic parameters. [ABSTRACT FROM AUTHOR]

Reverse transcriptases (RTs) are enzymes with DNA polymerase and RNase H activities. They convert ssRNA into dsDNA and are key enzymes for the replication of retroviruses and retroelements. Caulimoviridae is a major family of plant-infecting viruses. Caulimoviruses have a circular dsDNA genome that is replicated by reverse transcription, but in contrast to retroviruses, they lack integrase. Caulimoviruses are related to Ty3 retroelements. Ty3 RT has been extensively studied structurally and biochemically, but corresponding information for caulimoviral RTs is unavailable. In the present study, we report the first crystal structure of cauliflower mosaic virus (CaMV) RT in complex with a duplex made of RNA and DNA strands (RNA/DNA hybrid). CaMV RT forms a monomeric complex with the hybrid, unlike Ty3 RT, which does so as a dimer. Results of the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activity assays showed that individual CaMV RT molecules are able to perform full polymerase functions. However, our analyses showed that an additional CaMV RT molecule needs to transiently associate with a polymerase-competent RT molecule to execute RNase H cuts of the RNA strand. Collectively, our results provide details into the structure and function of CaMV RT and describe how the enzyme compares to other related RTs. [ABSTRACT FROM AUTHOR]

Endogenous pararetrovirus sequences (EPRVs) originated from DNA viruses of the family Caulimoviridae are widely present in plant genomes. Banana streak viruses (BSVs) are a group of circular double-stranded DNA viruses in the genus Badnavirus of the family Caulimoviridae. Banana endogenous virus sequences (BEVs) derived from the ancestral genes of badnaviruses and fixed in the genomes of various bananas. However, the genomic characteristics of BEVs remain unknown. In this study, we identified 2 new variants of BEVs GZ5 and GZ13 by sequences analyses, Southern blot, and fluorescent in situ hybridization (FISH). BEV GZ5 had one copy of integration in the BB genome of bananas, while BEV GZ13 was only present in the genome of the variety Dajiao. Importantly, BEV GZ5 contained a complete gene of reverse transcriptase (RT) and ribonuclease H (RNase H) (RT/RNase H). In addition, a 340-bp inverted repeat sequence partially overlapping with RNase H was found upstream and downstream of BEV GZ5. However, the amino acid sequences of BEV GZ5 had deletions and mutations compared with BSVs. The bioinformatics analyses showed that BEV GZ5 protein composed of 412 amino acids with a molecular weight of 47.37 kDa and an isoelectric point of 9.40. Leucine, isoleucine, and lysine (Lys) were the main amino acids of BEV GZ5 protein. The analyses revealed that BEV GZ5 protein contained 35 potential phosphorylation sites. Additionally, it was a hydrophilic protein without a signal peptide and transmembrane region. The secondary structure of BEV GZ5 protein consisted of 37.26% α-helix, followed by 36.25% random coil. To our knowledge, this is the first report that novel BEVs with the complete gene of RT/RNase H has been characterized, which provide a basis for further exploration the function and integration mechanism of BEVs in bananas. [ABSTRACT FROM AUTHOR]

RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance. The RNA helicase DHX9 is important for genome stability. Here the authors reveal that SUMOylation of DHX9 enhances its interaction with multiple proteins involved in the removal of harmful R-loop structures, thereby preventing DNA damage and cell death. [ABSTRACT FROM AUTHOR]

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson–Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO–PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs. [ABSTRACT FROM AUTHOR]

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure–activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) have been developed as potential therapeutic agents for genetic disorders and various life‐threatening conditions. This class of therapeutics facilitates the treatment of the currently undruggable targets. However, their low stability and poor activity must be overcome by developing new analogs with improved functions. Therefore, in this study, we synthesized 4′‐C‐(S)‐ and 4′‐C‐(R)‐2‐aminopropoxy−thymidine and investigated the stability of the modified oligonucleotides containing these analogs. Interestingly, both analogs responded differently to thermal denaturation, with the former exhibiting good stability closer to that of the unmodified oligonucleotide and the latter exhibiting slight thermal destabilization. Notably, DNA/RNA heteroduplexes modified with these analogs elicited RNase H activity by degrading the heteroduplexes upon treatment. In addition, the serum stability of the modified oligodeoxynucleotides was significantly enhanced by the introduction of these analogs. Overall, our findings highlight the potential application of these analogs as ASO therapeutic candidates. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low μM range (1.1–7.7 μM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure–activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium Wolbachia as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80-90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and Wolbachia from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from Brugia malayi containing the Wolbachia symbiont (wBm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5'-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of Wolbachia mRNA reads, resulting in a 16-1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the Brugia malayi depletion probes can remove rRNA from the filarial worm Dirofilaria immitis and the majority of rRNA from the more distantly related free living nematode Caenorhabditis elegans. These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library. [ABSTRACT FROM AUTHOR]

Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg2+ concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired. [ABSTRACT FROM AUTHOR]

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 μM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors. [ABSTRACT FROM AUTHOR]

Background: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. Methods: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. Results: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. Conclusions: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B. [ABSTRACT FROM AUTHOR]

High metastatic propensity of osteosarcoma leads to its therapeutic failure and poor prognosis. Although nuclear activation miRNAs (NamiRNAs) are reported to activate gene transcription via targeting enhancer and further promote tumor metastasis, it remains uncertain whether NamiRNAs regulate osteosarcoma metastasis and their exact mechanism. Here, we found that extracellular vesicles of the malignant osteosarcoma cells (143B) remarkably increased the migratory abilities of MNNG cells representing the benign osteosarcoma cells by two folds, which attributed to their high miR‐1246 levels. Specially, miR‐1246 located in nucleus could activate the migration gene expression (such as MMP1) to accelerate MNNG cell migration through elevating the enhancer activities via increasing H3K27ac enrichment. Instead, MMP1 expression was dramatically inhibited after Argonaute 2 (AGO2) knockdown. Notably, in vitro assays demonstrated that AGO2 recognized the hybrids of miR‐1246 and its enhancer DNA via PAZ domains to prevent their degradation from RNase H and these protective roles of AGO2 may favor the gene activation by miR‐1246 in vivo. Collectively, our findings suggest that miR‐1246 could facilitate osteosarcoma metastasis through interacting with enhancer to activate gene expression dependent on AGO2, highlighting the nuclear AGO2 as a guardian for NamiRNA‐targeted gene activation and the potential of miR‐1246 for osteosarcoma metastasis therapy. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system is promising for the reversal of tumor immunosuppression in immunotherapy, but the controlled delivery of CRISPR/Cas9 remains challenging. Herein, the study reported a smart DNA nanoassembly containing multivalent aptamers, realizing the controlled delivery of Cas9/sgRNA ribonucleoprotein (RNP) for enhanced cancer immunotherapy. A single‐stranded DNA complementary to sgRNA in the Cas9/sgRNA RNP can initiate a cascade‐clamped hybridization chain reaction (C‐HCR) to wrap the Cas9/sgRNA RNP up in the DNA nanoassembly. After selective internalization of DNA nanoassembly by cancer cells, Cas9/sgRNA RNP is released to cytoplasm in response to endogenous RNase H and enters the nuclei to knock out β‐catenin. The expression of the programmed death‐ligand one gene is effectively suppressed, and the immunosuppressive tumor microenvironment is reprogrammed. Meanwhile, the migration of cancer cells is inhibited, and the apoptosis of cancer cells is promoted. In a breast cancer mouse model, the administration of DNA nanoassembly effectively increased the infiltration of CD8+ T cells, eventually achieving high therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods. [ABSTRACT FROM AUTHOR]

This article discusses a case study of a 3-year-old girl with Aicardi-Goutières Syndrome (AGS) and rapidly progressive moyamoya vasculopathy (MMV). AGS is a genetic disorder characterized by increased production of interferon (IFN)-α2. Individuals with AGS often experience neurological impairments and are at a higher risk of stroke caused by MMV. The patient in this case study showed clinical improvement after treatment with baricitinib, a JAK1/2 inhibitor. The authors suggest that JAK inhibitors may have potential in stabilizing MMV in patients with AGS. [Extracted from the article]

Flooding stress, which reduces plant growth and seed yield, is a serious problem for soybean. To improve the productivity of flooded soybean, flooding-tolerant soybean was produced by gamma-ray irradiation. Three-day-old wild-type and mutant-line plants were flooded for 2 days. Protein, RNA, and genomic DNA were then analyzed based on oppositely changed proteins between the wild type and the mutant line under flooding stress. They were associated with cell organization, RNA metabolism, and protein degradation according to proteomic analysis. Immunoblot analysis confirmed that the accumulation of beta-tubulin/beta-actin increased in the wild type under flooding stress and recovered to the control level in the mutant line; however, alpha-tubulin increased in both the wild type and the mutant line under stress. Ubiquitin was accumulated and genomic DNA was degraded by flooding stress in the wild type; however, they were almost the same as control levels in the mutant line. On the other hand, the gene expression level of RNase H and 60S ribosomal protein did not change in either the wild type or the mutant line under flooding stress. Furthermore, chlorophyll a/b decreased and increased in the wild type and the mutant line, respectively, under flooding stress. These results suggest that the regulation of cell organization and protein degradation might be an important factor in the acquisition of flooding tolerance in soybean. [ABSTRACT FROM AUTHOR]

Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all AGOs retain a PIWI domain that takes an RNase H fold, the ancestor was likely an endoribonuclease (i.e., a slicer). In animals, most miRNA-mediated gene silencing occurs slicer independently. However, the slicer activity of AGO is indispensable in specific events, such as development and differentiation, which are critical for vertebrates and thus cannot be replaced by the slicer-independent regulation. This review highlights the distinctions in catalytic activation mechanisms among slicingcompetent AGOs, shedding light on the roles of two metal ions in target recognition and cleavage. The precision of the target specificity by the RNA-induced silencing complexes is reevaluated and redefined. The possible coevolutionary relationship between slicer-independent gene regulation and AGO-binding protein, GW182, is also explored. These discussions reveal that numerous captivating questions remain unanswered regarding the timing and manner in which AGOs employ their slicing activity. [ABSTRACT FROM AUTHOR]

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami‐based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM‐rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM‐guided assembly and pre‐designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo‐loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor‐associated gene for gene therapy in vivo. This rationally developed DNA origami‐based gene editing system presents a new avenue for the development of gene therapy. [ABSTRACT FROM AUTHOR]

R‐loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H‐deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA‐coated single‐stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU‐arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H. Synopsis: How R‐loops cause replication stress is incompletely understood. Here, toxic RNA:DNA hybrids are found to not only act as roadblocks for replication fork progression, but also interfere with stalled fork restart. RNA:DNA hybrids accumulate upon HU‐/MMS‐induced replication stress in yeast and human cells lacking RNases H activities.RNases H promote stalled fork resection, RPA‐ssDNA formation, and fork restart.RNA:DNA hybrids prevent postreplicative fork repair mechanisms in a transcription‐dependent manner. [ABSTRACT FROM AUTHOR]

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation, and several RNA-dependent ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement, and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 may have an expanded role in maintaining splicing fidelity.

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure–activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low μM range (1.1–7.7 μM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure–activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.

CRISPR‐Cas9 has demonstrated potential in inhibiting tumor growth by downregulating target gene expression, and the efficiency is expected to be enhanced with other nucleic acid drugs. Herein, a dual‐enzyme–responsive DNA‐based nanoframework (NF) that enables controlled co‐delivery of Cas9 ribonucleoprotein (RNP) and antisense oligodeoxynucleotide (ASO) for synergistic gene therapy is reported. NF is synthesized with acrylamide DNA (acDNA) as a cross‐linker and loading site. A ternary complex containing sgRNA, ASO, and Cas9 that bound to acDNA is designed. The phase transition property of NF allows to reversibly swell and aggregate thermo‐responsively for the high‐capacity loading of ternary complex. RNA is cleaved from DNA–RNA complex by the overexpressed ribonuclease H for the controlled release of Cas9 RNP, and the bifurcated DNA is cleaved by the overexpressed flap structure‐specific endonuclease 1 for the controlled release of overhanging ASO. The combination of gene editing with Cas9 RNP and gene silencing with ASO allows simultaneous manipulation of target gene in nucleus and its mRNA in cytoplasm, and this synergistic gene therapy has shown remarkable therapeutic effect in a breast cancer mouse model, making it a promising therapeutic strategy for future cancer treatment. [ABSTRACT FROM AUTHOR]

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross‐sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h‐motif structure incorporating double‐crossover (DX) tile‐like DNA edges to achieve structural rigidity and provide efficient self‐assembly of h‐motif‐based DNA nanotube (H‐NT) units, thus producing programmable, micrometer‐long nanotubes. We demonstrate control of the H‐NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H‐NTs can assemble at near‐physiological salt conditions and will serve as an easily synthesized, DNA‐economical modular template for biosensors, plasmonics, or other functional materials and as cost‐efficient drug‐delivery vehicles for biomedical applications. [ABSTRACT FROM AUTHOR]

Issues surrounding rapid degradation and limited therapeutic efficacy still exist in the development of native antisense oligonucleotides (ASONs). In this paper, a novel strategy of chimeric 4A2–5-ASON prodrug combined with chemotherapy for oncotherapy was proposed. The self-assembled hairpin-end prodrug structure provided a DOX loading site, while enhancing stability against nuclease degradation. The disulfide led responsive drug release, and excellent therapeutic effects were achieved by the combined action of RNase H and RNase L recruitment, along with chemotherapy drug Doxorubicin (DOX), both in vitro and in vivo. This work provides evidence for the development of designing nucleic acid drugs with combined mechanisms. [ABSTRACT FROM AUTHOR]

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3′ untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections. [ABSTRACT FROM AUTHOR]

The article focuses on mechanosensation in plant leaf veins, highlighting whether the plant vasculature can sense touch. Topics include the development of a quantitative assay to investigate touch-response electrical signals in leaves and veins of Arabidopsis thaliana, the role of various genes, and the discovery that primary veins have autonomous mechanosensory properties.

Retroviruses originated from long terminal repeat retrotransposons (LTR-RTs) through several structural adaptations. One such modification was the arrangement of an additional ribonuclease H (aRH) domain next to native RH, followed by degradation and subfunctionalization of the latter. We previously showed that this retrovirus-like structure independently evolved in Tat LTR-RTs in flowering plants, proposing its origin from sequential rearrangements of ancestral Tat structures identified in lycophytes and conifers. However, most nonflowering plant genome assemblies were not available at that time, therefore masking the history of aRH acquisition by Tat and challenging our hypothesis. Here, we revisited Tat's evolution scenario upon the aRH acquisition by covering most of the extant plant phyla. We show that Tat evolved and obtained aRH in an ancestor of land plants. Importantly, we found the retrovirus-like structure in clubmosses, hornworts, ferns, and gymnosperms, suggesting its ancient origin, broad propagation, and yet-to-be-understood benefit for the LTR-RTs' adaptation. [ABSTRACT FROM AUTHOR]

The genomes of mammals contain fingerprints of past infections by ancient retroviruses that invaded the germline of their ancestors. Most of these endogenous retroviruses (ERVs) contain only remnants of the original retrovirus; however, on rare occasions, ERV genes can be co-opted for a beneficial host function. While most studies of co-opted ERVs have focused on envelope genes, including the syncytins that function in placentation, there are examples of co-opted gag genes including one we recently discovered in simian primates. Here, we searched for other intact gag genes in nonprimate mammalian lineages. We began by examining the genomes of extant camel species, which represent a basal lineage in the order Artiodactyla. This identified a gagpol gene with a large open reading frame (ORF) (>3,500 bp) in the same orthologous location in Artiodactyla species but that is absent in other mammals. Thus, this ERV was fixed in the common ancestor of all Artiodactyla at least 64 million years ago. The amino acid sequence of this gene, termed ARTgagpol, contains recognizable matrix, capsid, nucleocapsid, and reverse transcriptase domains in ruminants, with an RNase H domain in camels and pigs. Phylogenetic analysis and structural prediction of its reverse transcriptase and RNase H domains groups ARTgagpol with gammaretroviruses. Transcriptomic analysis shows ARTgagpol expression in multiple tissues suggestive of a co-opted host function. These findings identify the oldest and largest ERV-derived gagpol gene with an intact ORF in mammals, an intriguing milestone in the co-evolution of mammals and retroviruses. IMPORTANCE: Retroviruses are unique among viruses that infect animals as they integrate their reverse-transcribed double-stranded DNA into host chromosomes. When this happens in a germline cell, such as sperm, egg, or their precursors, the integrated retroviral copies can be passed on to the next generation as endogenous retroviruses (ERVs). On rare occasions, the genes of these ERVs can be domesticated by the host. In this study we used computational similarity searches to identify an ancient ERV with an intact viral gagpol gene in the genomes of camels that is also found in the same genomic location in other even-toed ungulates suggesting that it is at least 64 million years old. Broad tissue expression and predicted preservation of the reverse transcriptase fold of this protein suggest that it may be domesticated for a host function. This is the oldest known intact gagpol gene of an ancient retrovirus in mammals. [ABSTRACT FROM AUTHOR]

Although the temporary presence of ribonucleotides in DNA is normal, their persistence represents a form of DNA damage. Here, we assess such damage and damage defense to DNA in plastids and mitochondria of maize. Shoot development proceeds from meristematic, non-pigmented cells containing proplastids and promitochondria at the leaf base to non-dividing green cells in the leaf blade containing mature organelles. The organellar DNAs (orgDNAs) become fragmented during this transition. Previously, orgDNA damage and damage defense of two types, oxidative and glycation, was described in maize, and now a third type, ribonucleotide damage, is reported. We hypothesized that ribonucleotide damage changes during leaf development and could contribute to the demise of orgDNAs. The levels of ribonucleotides and R-loops in orgDNAs and of RNase H proteins in organelles were measured throughout leaf development and in leaves grown in light and dark conditions. The data reveal that ribonucleotide damage to orgDNAs increased by about 2- to 5-fold during normal maize development from basal meristem to green leaf and when leaves were grown in normal light conditions compared to in the dark. During this developmental transition, the levels of the major agent of defense, RNase H, declined. The decline in organellar genome integrity during maize development may be attributed to oxidative, glycation, and ribonucleotide damages that are not repaired. [ABSTRACT FROM AUTHOR]

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.

In this work we investigated, for the first time, the effect of Plagius flosculosus (L.) Alavi & Heywood, a Sardinian–Corsican endemic plant, on HIV-1 integrase (IN) activity. The phytochemical analysis of the leaves chloroform extract led us to isolate and characterize three compounds (SPK1, SPK2, and SPK3) belonging to the spiroketals, a group of naturally occurring metabolites of phytochemical relevance with interesting biological properties. Due to their structural diversity, these cyclic ketals have attracted the interest of chemists and biologists. SPK1, SPK2, and SPK3 were evaluated here for their ability to inhibit HIV-1 integrase activity in biochemical assays. The results showed that all the compounds inhibited HIV-1 IN activity. In particular, the most active one was SPK3, which interfered in a low molecular range (IC50 of 1.46 ± 0.16 µM) with HIV-1 IN activity in the presence/absence of the LEDGF cellular cofactor. To investigate the mechanism of action, the three spiroketals were also tested on HIV-1 RT-associated Ribonuclease H (RNase H) activity, proving to be active in inhibiting this function. Although SPK3 was unable to inhibit viral replication in cell culture, it promoted the IN multimerization. We hypothesize that SPK3 inhibited HIV-1 IN through an allosteric mechanism of action. [ABSTRACT FROM AUTHOR]

This study examines the contributions of ribonucleases (RNase)and human genes to hybrid removal, mutagenesis, and DNA replication genome-wide, revealing distinct patterns of mutagenesis and hybrid accumulation in cells lacking these ribonucleases. The accumulation of hybrids is found to be influenced by gene expression levels, and disruptions in DNA replication in certain cells lead to transversions and structural variations.

Chemically modified antisense oligonucleotides (ASO) currently in pre‐clinical and clinical experiments mainly focus on the 2′‐position derivatizations to enhance stability and targeting affinity. Considering the possible incompatibility of 2′‐modifications with RNase H stimulation and activity, we have hypothesized that the atom specific modifications on nucleobases can retain the complex structure and RNase H activity, while enhancing ASO's binding affinity, specificity, and stability against nucleases. Herein we report a novel strategy to explore our hypothesis by synthesizing the deoxynucleoside phosphoramidite building block with the seleno‐modification at 5‐position of thymidine, as well as its Se‐oligonucleotides. Via X‐ray crystal structural study, we found that the Se‐modification was located in the major groove of nucleic acid duplex and didn't cause the thermal and structural perturbations. Surprisingly, our nucleobase‐modified Se‐DNAs were exceptionally resistant to nuclease digestion, while compatible with RNase H activity. This affords a novel avenue for potential anti‐sense modification in the form of Se‐antisense oligonucleotides (Se‐ASO). [ABSTRACT FROM AUTHOR]

Grass pea is a promising crop with the potential to provide food and fodder, but its genomics has not been adequately explored. Identifying genes for desirable traits, such as drought tolerance and disease resistance, is critical for improving the plant. Grass pea currently lacks known R-genes, including the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family, which plays a key role in protecting the plant from biotic and abiotic stresses. In our study, we used the recently published grass pea genome and available transcriptomic data to identify 274 NBS-LRR genes. The evolutionary relationships between the classified genes on the reported plants and LsNBS revealed that 124 genes have TNL domains, while 150 genes have CNL domains. All genes contained exons, ranging from 1 to 7. Ten conserved motifs with lengths ranging from 16 to 30 amino acids were identified. We found TIR-domain-containing genes in 132 LsNBSs, with 63 TIR-1 and 69 TIR-2, and RX-CCLike in 84 LsNBSs. We also identified several popular motifs, including P-loop, Uup, kinase-GTPase, ABC, ChvD, CDC6, Rnase_H, Smc, CDC48, and SpoVK. According to the gene enrichment analysis, the identified genes undergo several biological processes such as plant defense, innate immunity, hydrolase activity, and DNA binding. In the upstream regions, 103 transcription factors were identified that govern the transcription of nearby genes affecting the plant excretion of salicylic acid, methyl jasmonate, ethylene, and abscisic acid. According to RNA-Seq expression analysis, 85% of the encoded genes have high expression levels. Nine LsNBS genes were selected for qPCR under salt stress conditions. The majority of the genes showed upregulation at 50 and 200 µM NaCl. However, LsNBS-D18, LsNBS-D204, and LsNBS-D180 showed reduced or drastic downregulation compared to their respective expression levels, providing further insights into the potential functions of LsNBSs under salt stress conditions. They provide valuable insights into the potential functions of LsNBSs under salt stress conditions. Our findings also shed light on the evolution and classification of NBS-LRR genes in legumes, highlighting the potential of grass pea. Further research could focus on the functional analysis of these genes, and their potential use in breeding programs to improve the salinity, drought, and disease resistance of this important crop. [ABSTRACT FROM AUTHOR]

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3′-to-5′ exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases. [ABSTRACT FROM AUTHOR]

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

Although the toxicity of triazophos is high and it has been pulled from the market in many countries; it is still widely used and frequently detected in agricultural products. While conventional analyses have been routinely used for the quantification and monitoring of triazophos residues, those for detecting low residual levels are deemed necessary. Therefore, we developed a novel and sensitive fluorometric signal amplification immunoassay employing bio-barcodes for the quantitative analysis of triazophos residues in foodstuffs and surface water. Herein, monoclonal antibodies (mAbs) attached to gold nanoparticles (AuNPs) were coated with DNA oligonucleotides (used as a signal generator), and a complementary fluorogenic RNA was used for signal amplification. The system generated detection signals through DNA-RNA hybridization and subsequent dissociation of fluorophores by Ribonuclease H (RNase H). It has to be noted that RNase H can only disintegrate the RNA in DNA-RNA duplex, but not cleave single or double-stranded DNA. Hence, with iterative cycles of DNA-RNA hybridization, sufficient strong signal was obtained for reliable detection of residues. Furthermore, this method enables quantitative detection of triazophos residues through fluorescence intensity measurements. The competitive immunoassay shows a wide linear range of 0.01-100 ng/mL with a limit of detection (LOD) of 0.0032 ng/mL. The assay substantially meets the demand for the low residue detection of triazophos residues in agricultural products and water samples. Accuracy (expressed as spiked recovery %) and coefficient of variation (CV) were ranged from 73.4% to 116% and 7.04% to 17.4%, respectively. The proposed bio-barcodes immunoassay has the advantages of being stable, reproducible, and reliable for residue detection. In sum, the present study provides a novel approach for detection of small molecules in various sample matrices.

The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5′ RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5′-3′ flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5′ single flap, and nicked RNA-DNA hybrid substrates. We show that the 5′ nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5′ flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5′ nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5′ flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human. Author summary: Proteins with 5′ flap endo/exonuclease (FEN) activity provide an essential contribution to DNA replication and repair in all cellular life. In bacteria, DNA polymerase I is thought to be the central enzyme involved in Okazaki fragment processing, using its DNA polymerase and 5′ nuclease activities to generate and then remove the 5′ ssRNA segment of an Okazaki fragment. Many bacterial genomes encode a second, discrete FEN in addition to Pol I. We show that FEN is the primary 5′ nuclease used by B. subtilis for primer removal. FEN activity exceeds that of Pol I on most substrates, including several that mimic Okazaki fragment intermediates. Additionally, we provide genetic evidence showing that FEN is involved in Okazaki fragment processing and that it is the DNA polymerase domain of Pol I rather than its 5′ nuclease domain that is important in vivo. With our results, we propose a new model for Okazaki fragment processing in B. subtilis, which may be prevalent in a wider group of bacteria than previously appreciated. [ABSTRACT FROM AUTHOR]

Controlling cell-free expression of a gene to protein with non-invasive stimuli is vital to the future application of DNA nanodevices and synthetic cells. However, little emphasis has been placed on developing light-controlled 'off' switches for cell-free expression. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells; however, they are complicated to synthesise and have not been tested in cell-free systems. Developing simple, accessible methods to produce light-activated antisense oligonucleotides will be crucial for allowing their application in cell-free biology and biotechnology. Here, we report a mild, one-step method for selectively attaching commercially-available photoremovable protecting groups, photocages, onto phosphorothioate linkages of antisense oligonucleotides. Using this photocaging method, upon illumination, the original phosphorothioate antisense oligonucleotide is reformed. Photocaged antisense oligonucleotides, containing mixed phosphorothioate and phosphate backbones, showed a drastic reduction in duplex formation and RNase H activity, which was recovered upon illumination. We then demonstrated that these photocaged antisense oligonucleotides can be used to knock down cell-free protein synthesis using light. This simple and accessible technology will have future applications in light-controlled biological logic gates and regulating the activity of synthetic cells. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells, however, their synthesis remains challenging and application in cell-free systems is underexplored. Here, the authors report a one-step method for selectively attaching photocages onto phosphorothioate linkages of antisense oligonucleotides that can knockdown cell-free protein synthesis using light. [ABSTRACT FROM AUTHOR]

In the present work, a novel approach was developed for the preparation of Cu2O nanoparticle decorated MnO2 nanosheets (Cu2O@MnO2). Uniformly dispersed Cu2O nanocrystals were produced on the surface of MnO2 nanosheets by in situ reduction under refluxing conditions. The unique structure of the used MnO2 nanosheet support played a vital role in the preparation of such Cu2O@MnO2 nanocomposites. The electrogenerated chemiluminescence (ECL) resonance energy transfer can occur between the luminol/H2O2 system and Cu2O@MnO2 nanocomposites, resulting in a decrease of the ECL intensity, which can be used to fabricate an ECL sensor. Cu2O@MnO2 nanocomposite modified heterologous DNA/RNA duplexes were modified on the GCE to construct an ECL-RET system, leading to the decrease of ECL intensity. As a highly conserved damage repair protein, RNase H can specifically hydrolyze RNA in DNA/RNA strands to release Cu2O@MnO2 nanocomposites and recover the ECL signal. As a result, an "off–on" mode ECL sensor for sensitive RNase H assay was fabricated. Under the optimal conditions, the detection limit of RNase H is 0.0005 U mL−1, which is superior to other approaches. The proposed method provides a universal platform for monitoring RNase H, and exhibits great potential in bioanalysis. [ABSTRACT FROM AUTHOR]