Search Results (137)

Contact unmodified antisense DNA biotechnology (CUADb), developed in 2008, employs short antisense DNA oligonucleotides (oligos) as a novel approach to insect pest control. These oligonucleotide-based insecticides target pest mature rRNAs and/or pre-rRNAs and have demonstrated high insecticidal efficacy, particularly against sap-feeding insect pests, which are key vectors of plant DNA viruses and among the most economically damaging herbivorous insects. To further explore the potential of CUADb, this study evaluated the insecticidal efficacy of short 11-mer antisense DNA oligos against Coccus hesperidum, in comparison with long 56-mer single-stranded and double-stranded DNA sequences. The short oligos exhibited higher insecticidal activity. By day 9, the highest mortality rate (97.66 ± 4.04%) was recorded in the Coccus-11 group, while the most effective long sequence was the double-stranded DNA in the dsCoccus-56 group (77.09 ± 6.24%). This study also describes the architecture of the DNA containment (DNAc) mechanism, highlighting the intricate interactions between rRNAs and various types of DNA oligos. During DNAc, the Coccus-11 treatment induced enhanced ribosome biogenesis and ATP production through a metabolic shift from carbohydrates to lipid-based energy synthesis. However, this ultimately led to a ‘kinase disaster’ due to widespread kinase downregulation resulting from insufficient ATP levels. All DNA oligos with high or moderate complementarity to target rRNA initiated hypercompensation, but subsequent substantial rRNA degradation and insect mortality occurred only when the oligo sequence perfectly matched the rRNA. Both short and long oligonucleotide insecticide treatments led to a 3.75–4.25-fold decrease in rRNA levels following hypercompensation, which was likely mediated by a DNA-guided rRNase, such as RNase H1, while crucial enzymes of RNAi (DICER1, Argonaute 2, and DROSHA) were downregulated, indicating fundamental difference in molecular mechanisms of DNAc and RNAi. Consistently, significant upregulation of RNase H1 was detected in the Coccus-11 treatment group. In contrast, treatment with random DNA oligos resulted in only a 2–3-fold rRNA decrease, consistent with the normal rRNA half-life maintained by general ribonucleases. These findings reveal a fundamental new mechanism of rRNA regulation via complementary binding between exogenous unmodified antisense DNA and cellular rRNA. From a practical perspective, this minimalist approach, applying short antisense DNA dissolved in water, offers an effective, eco-friendly and innovative solution for managing sternorrhynchans and other insect pests. The results introduce a promising new concept in crop protection: DNA-programmable insect pest control. Full article

The potent and selective ‘genetic zipper’ method for insect pest control consists of three essential components: an antisense DNA (the finder), its complementary mature rRNA or pre-rRNA of the pest (the target), and the host’s endogenous DNA-guided rRNase (the degrader). Although this approach has been validated, the spectrum of effective rRNA targets remains insufficiently explored. In this study, we report for the first time the insecticidal efficacy of a novel oligonucleotide insecticide, Eriola-11, which targets the mitochondrial 16S rRNA of the woolly apple aphid Eriosoma lanigerum Hausmann. We hypothesized that the antisense-mediated silencing of mitochondrial rRNA would impair aphid viability and lead to physiological disruptions associated with mitochondrial energy metabolism. Eriola-11 was applied either once or twice (with a 24 h interval) to aphid-infested plants, and aphid mortality was recorded over 14 days. Mitochondrial 16S rRNA expression levels were quantified using molecular assays, and the degradation kinetics of Eriola-11 were assessed in aphid tissue homogenates. Results showed significant insecticidal activity, with 67.55% mortality after a single treatment and 83.35% after two treatments. Treated aphids exhibited the loss of their characteristic white woolly wax covering, and mitochondrial 16S rRNA expression was reduced 0.66-fold relative to the control. Additionally, Eriola-11 was fully degraded by aphid DNases from tissue homogenates within 3 h, highlighting its rapid biodegradability. These findings establish mitochondrial 16S rRNA as a viable target for antisense insecticides and expand the catalogue of potential rRNA-based targets, offering a promising avenue for environmentally sustainable pest control strategies. Full article

The selective regulation of gene expression at the RNA level represents a rapidly evolving field offering substantial clinical potential. This review examines the molecular mechanisms of intracellular enzymatic systems that utilize single-stranded nucleic acids to downregulate specific RNA targets. The analysis encompasses antisense oligonucleotides and synthetic mimics of small interfering RNA (siRNA), microRNA (miRNA), transfer RNA-derived small RNA (tsRNA), and PIWI-interacting RNA (piRNA), elucidating their intricate interactions with crucial cellular machinery, specifically RNase H1, RNase P, AGO, and PIWI proteins, mediating their biological effects. The functional and structural characteristics of these endonucleases are examined in relation to their mechanisms of action and resultant therapeutic outcomes. This comprehensive analysis illuminates the interactions between single-stranded nucleic acids and their endonuclease partners, covering antisense inhibition pathways as well as RNA interference processes. This field of research has important implications for advancing targeted RNA modulation strategies across various disease contexts. Full article

Targeting ribonuclease H (RNase H) has emerged as a highly promising strategy for treating HIV-1. In this study, a series of novel 3-hydrazonoindolin-2-one derivatives were designed and synthesized as potential inhibitors of HIV-1 RNase H. Notably, several of these derivatives displayed micromolar inhibitory activity. Among the compounds examined, the hit compound demonstrated potent inhibition of HIV-1 RNase H, boasting a Ki value of 2.31 μM. Additionally, the most potent compound of this general structure exhibited remarkable inhibitory activity, with Ki values of 0.55 μM. Through docking studies, the key interactions of this ligand within the active site of RNase H were uncovered. This novel chemical structure can be regarded as a prospective scaffold for the future development of RNase H inhibitors. Full article

Background: HIV-1 RT inhibitors were the first drugs approved to treat AIDS and remain key components of highly active antiretroviral therapy (HAART). While HAART effectively suppresses viral replication and slows disease progression, it has limitations, including long-term side effects and the emergence of drug-resistant strains, highlighting the need for new treatments. Objectives: Based on our previous experience, and insights from existing inhibitors of HIV-1 RT and RNase H, we aim to design and synthesize safer, multifunctional molecules. Methods: Using molecular docking studies, these compounds will incorporate pharmacophores targeting multiple stages of the HIV life cycle to enhance efficacy, reduce resistance, and improve pharmacokinetics. The compounds were synthesized via a one-pot three component reaction. The synthesized compounds were identified using spectroscopy and tested in vitro for activity against key HIV targets, including RNA-dependent DNA polymerase (RDDP) and RNAse H. Results: Among the synthesized compounds, several demonstrated strong inhibitory activity, with compound 11 showing IC₅₀ values comparable to the reference drug Nevirapine, and compound 4 exhibiting dual inhibition of both RT and RNase H activities. Conclusions: These findings emphasize the importance of a multidisciplinary approach, combining computational modeling with experimental validation, to identify promising leads for therapeutic development. Full article

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4′-C-substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4′-C-(N-ethyl) aminoethyl (4′-EAE-T), 4′-C-(N-butyl) aminoethyl (4′-BAE-T), and 4′-C-(N-octyl) aminoethyl (4′-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4′-C-aminoethyl (4′-AE-T) and 4′-C-(N-methyl) aminoethyl (4′-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4′-EAE-T and 4′-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4′-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4′-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4′-EAE-T resulting in the highest stability, followed by 4′-BAE-T and 4′-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4′-OAE-modified nucleotide, which exhibited low activity. Full article

There is a strong demand for new and efficient antiviral compounds. A series of 2-hydroxy-1,4-naphthoquinone Mannich bases were screened for their HIV-1-RNase H inhibitory activity. An HIV-1-RNase H assay was used to study the RNase H inhibition by the test compounds. Docking of active derivatives into the active site of the enzyme was carried out. Compounds 1e and 2k showed distinctly higher HIV-1-RNase H inhibitory activity (IC₅₀ = 2.8–3.1 µM) than the known inhibitors RDS1759 and compound 13. The binding mode and possible interactions of 1e and 2k with the HIV-1-RNase H active site were determined using molecular docking, which led to the identification of salient and concealed pharmacophoric features of these molecules. The docking analysis revealed that there are significant differences in the binding mode of these compounds within the active site of the target enzyme. A selection of HIV-1-RNase H-inhibitory Mannich bases was tested for antiviral activity against HIV-1, and compound 2k showed the highest activity at low toxicity to host cells. The lawsone Mannich bases 1e and 2k also underwent a preliminary screening for activity against SARS-CoV-2, and compound 1e was found to inhibit SARS-CoV-2 replication (IC₅₀ = 11.2 µM). Full article

The potential role of reactive oxygen species (ROS) is studied in the male gametophytes of petunia (Petunia hybrida E. Vilm.) grown in vivo with a focus on its germination, growth support in the progamic stage of fertilization, and the function of the mechanism underlying S-RNase-based self-incompatibility. Exogenous treatment with H₂O₂ influences the in vivo germination and polar growth of pollen tubes (PTs), which manifests as the acceleration or inhibition of these processes depending on its concentration, time interval after pollination, and pollination variant. The H₂O₂ treatment of the stigma somewhat stimulates the PT elongation in the late stages of self-incompatible pollination (4–8 h) versus the strong PT inhibition observed during the first hour of germination. A different pattern is observable in cross-compatible pollination: the H₂O₂ treatment of pistils inhibits PT growth during the overall pollination at all tested concentrations. Treatment of pistils with the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) strongly inhibited the growth of PTs in both pollination variants. In addition, DCF-DA staining confirms that ROS are formed in pollen, PTs, stigma of nonpollinated pistil, and the pistil itself in all pollination variants. The PT growth during the function of the self-incompatibility mechanism is arrested at high ROS concentrations, which is presumably associated with the SI-induced programmed cell death. Our results demonstrate that ROS are a necessary component of pollen, PTs, exudate, and stigma cells and contribute to successful reproduction. This study provides a deeper insight into the ROS functions during the PT growth in an in vivo system. Full article

The development of new convenient tools for the design of multicomponent nucleic acid (NA) complexes is one of the challenges in biomedicine and NA nanotechnology. In this paper, we analyzed the formation of hybrid RNA/DNA concatemers and self-limited complexes by a pair of oligonucleotides using UV melting, circular dichroism spectroscopy, and a gel shift assay. Effects of the size of the linker between duplex-forming segments of the oligonucleotides on complexes’ shape and number of subunits were compared and systematized for RNA/DNA, DNA/DNA, and RNA/RNA assemblies. The data on complex types summarized here as heat maps offer a convenient tool for the design of NA constructs. General rules found for RNA/DNA, DNA/DNA, and RNA/RNA complexes allow not only designing complexes with desired structures but also purposefully transforming their geometry. The A-form of the double helix of the studied RNA/DNA complexes was confirmed by circular dichroism analysis. Moreover, we show for the first time efficient degradation of RNA in hybrid self-limited complexes by RNase H and imidazole. The results open up new prospects for the design of supramolecular complexes as tools for nanotechnology, nanomachinery, and biomedical applications. Full article

R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants K_d were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1. Full article

The secondary metabolites of seawater and freshwater blue-green algae are a rich natural product pool containing diverse compounds with various functions, including antiviral compounds; however, high-efficiency methods to screen such compounds are lacking. Advanced virtual screening techniques can significantly reduce the time and cost of novel antiviral drug identification. In this study, we used a cyanobacterial secondary metabolite library as an example and trained three models to identify compounds with potential antiviral activity using a machine learning method based on message-passing neural networks. Using this method, 364 potential antiviral compounds were screened from >2000 cyanobacterial secondary metabolites, with amides predominating (area under the receiver operating characteristic curve value: 0.98). To verify the actual effectiveness of the candidate antiviral compounds, HIV virus reverse transcriptase (HIV-1 RT) was selected as a target to evaluate their antiviral potential. Molecular docking experiments demonstrated that candidate compounds, including kororamide, mollamide E, nostopeptolide A3, anachelin-H, and kasumigamide, produced relatively robust non-covalent bonding interactions with the RNase H active site on HIV-1 RT, supporting the effectiveness of the proposed screening model. Our data demonstrate that artificial intelligence-based screening methods are effective tools for mining potential antiviral compounds, which can facilitate the exploration of various natural product libraries. Full article

Salinity causes widespread crop loss and prompts plants to adapt through changes in gene expression. In this study, we aimed to investigate the function of the non-tandem CCCH zinc-finger (non-TZF) protein gene AtC3H3 in response to salt stress in Arabidopsis. AtC3H3, a gene from the non-TZF gene family known for its RNA-binding and RNase activities, was up-regulated under osmotic stress, such as high salt and drought. When overexpressed in Arabidopsis, AtC3H3 improved tolerance to salt stress, but not drought stress. The expression of well-known abscisic acid (ABA)-dependent salt stress-responsive genes, namely Responsive to Desiccation 29B (RD29B), RD22, and Responsive to ABA 18 (RAB18), and representative ABA-independent salt stress-responsive genes, namely Dehydration-Responsive Element Binding protein 2A (DREB2A) and DREB2B, was significantly higher in AtC3H3-overexpressing transgenic plants (AtC3H3 OXs) than in wild-type plants (WT) under NaCl treatment, indicating its significance in both ABA-dependent and -independent signal transduction pathways. mRNA-sequencing (mRNA-Seq) analysis using NaCl-treated WT and AtC3H3 OXs revealed no potential target mRNAs for the RNase function of AtC3H3, suggesting that the potential targets of AtC3H3 might be noncoding RNAs and not mRNAs. Through this study, we conclusively demonstrated that AtC3H3 plays a crucial role in salt stress tolerance by influencing the expression of salt stress-responsive genes. These findings offer new insights into plant stress response mechanisms and suggest potential strategies for improving crop resilience to salinity stress. Full article

R-loops, structures that play a crucial role in various biological processes, are integral to gene expression, the maintenance of genome stability, and the formation of epigenomic signatures. When these R-loops are deregulated, they can contribute to the development of serious health conditions, including cancer and neurodegenerative diseases. The detection of R-loops is a complex process that involves several approaches. These include S9.6 antibody- or RNAse H-based immunoprecipitation, non-denaturing bisulfite footprinting, gel electrophoresis, and electron microscopy. Each of these methods offers unique insights into the nature and behavior of R-loops. In our study, we introduce a novel protocol that has been developed based on a single-molecule DNA combing assay. This innovative approach allows for the direct and simultaneous visualization of RNA:DNA hybrids and replication forks, providing a more comprehensive understanding of these structures. Our findings confirm the transcriptional origin of the hybrids, adding to the body of knowledge about their formation. Furthermore, we demonstrate that these hybrids have an inhibitory effect on the progression of replication forks, highlighting their potential impact on DNA replication and cellular function. Full article

Helicoverpa armigera is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for H. armigera have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species H. zea. Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of H. armigera. Full article

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. Full article