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RNA (New York, N.Y.) May 2024Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for... (Comparative Study)
Comparative Study
Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene upon delivery with a KFF-peptide carrier into Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.
Topics: Anti-Bacterial Agents; Peptide Nucleic Acids; Oligonucleotides, Antisense; Morpholinos; Peptides; Humans
PubMed: 38413166
DOI: 10.1261/rna.079969.124 -
Nucleic Acids Research Apr 2024The field of synthetic nucleic acids with novel backbone structures [xenobiotic nucleic acids (XNAs)] has flourished due to the increased importance of XNA antisense...
The field of synthetic nucleic acids with novel backbone structures [xenobiotic nucleic acids (XNAs)] has flourished due to the increased importance of XNA antisense oligonucleotides and aptamers in medicine, as well as the development of XNA processing enzymes and new XNA genetic materials. Molecular modeling on XNA structures can accelerate rational design in the field of XNAs as it contributes in understanding and predicting how changes in the sugar-phosphate backbone impact on the complementation properties of the nucleic acids. To support the development of novel XNA polymers, we present a first-in-class open-source program (Ducque) to build duplexes of nucleic acid analogs with customizable chemistry. A detailed procedure is described to extend the Ducque library with new user-defined XNA fragments using quantum mechanics (QM) and to generate QM-based force field parameters for molecular dynamics simulations within standard packages such as AMBER. The tool was used within a molecular modeling workflow to accurately reproduce a selection of experimental structures for nucleic acid duplexes with ribose-based as well as non-ribose-based nucleosides. Additionally, it was challenged to build duplexes of morpholino nucleic acids bound to complementary RNA sequences.
Topics: Molecular Dynamics Simulation; Morpholinos; Nucleic Acid Conformation; Nucleic Acids; Oligonucleotides; RNA; Software
PubMed: 38412249
DOI: 10.1093/nar/gkae135 -
The Journal of Biological Chemistry Apr 2024First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized...
First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.
Topics: Animals; Humans; Mice; Carrier Proteins; Cell Polarity; Dishevelled Proteins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nerve Tissue Proteins; Protein Biosynthesis; Protein Isoforms; Xenopus laevis; Xenopus Proteins; Zebrafish; Zebrafish Proteins
PubMed: 38403249
DOI: 10.1016/j.jbc.2024.106792 -
International Journal of Molecular... Feb 2024The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors...
The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, and genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, gene knockdown induced a greater decrease in cell proliferation, compared to gene knockdown and strong upregulation of and , as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.
Topics: Humans; Biomarkers; Cell Hypoxia; Cell Line, Tumor; Gene Knockdown Techniques; Glioblastoma; Hypoxia; Glucose Transporter Type 3
PubMed: 38396757
DOI: 10.3390/ijms25042079 -
STAR Protocols Mar 2024Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here,...
Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments. For complete details on the use and execution of this protocol, please refer to Kakebeen et al., Patel et al., and Patel et al..
Topics: Animals; Xenopus; Xenopus laevis; Larva
PubMed: 38367232
DOI: 10.1016/j.xpro.2024.102895 -
HGG Advances Apr 2024Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory...
Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory insufficiency, and are collectively known as desminopathies. While most DES pathogenic variants act via a dominant mechanism, recessively acting variants have also been reported. Currently, there are no effective therapeutic interventions for desminopathies of any type. Here, we report an affected individual with rapidly progressive dilated cardiomyopathy, requiring heart transplantation at age 13 years, in the setting of childhood-onset skeletal muscle weakness. We identified biallelic DES variants (c.640-13 T>A and c.1288+1 G>A) and show aberrant DES gene splicing in the affected individual's muscle. Through the generation of an inducible lentiviral system, we transdifferentiated fibroblast cultures derived from the affected individual into myoblasts and validated this system using RNA sequencing. We tested rationally designed, custom antisense oligonucleotides to screen for splice correction in these transdifferentiated cells and a functional minigene splicing assay. However, rather than correctly redirecting splicing, we found them to induce undesired exon skipping. Our results indicate that, while an individual precision-based molecular therapeutic approach to splice-altering pathogenic variants is promising, careful preclinical testing is imperative for each novel variant to test the feasibility of this type of approach for translation.
Topics: Adolescent; Humans; Cardiomyopathies; Cardiomyopathy, Dilated; Mutation; RNA Splicing
PubMed: 38358893
DOI: 10.1016/j.xhgg.2024.100274 -
Scientific Reports Feb 2024In contrast to adult mammals, zebrafish display a high capacity to heal injuries and repair damage to various organs. One of the earliest responses to injury in adult...
In contrast to adult mammals, zebrafish display a high capacity to heal injuries and repair damage to various organs. One of the earliest responses to injury in adult zebrafish is revascularization, followed by tissue morphogenesis. Tissue vascularization entails the formation of a blood vessel plexus that remodels into arteries and veins. The mechanisms that coordinate these processes during vessel regeneration are poorly understood. Hence, investigating and identifying the factors that promote revascularization and vessel remodeling have great therapeutic potential. Here, we revealed that fin vessel remodeling critically depends on Apela peptide. We found that Apela selectively accumulated in newly formed zebrafish fin tissue and vessels. The temporal expression of Apela, Apln, and their receptor Aplnr is different during the regenerative process. While morpholino-mediated knockdown of Apela (Mo-Apela) prevented vessel remodeling, exogenous Apela peptide mediated plexus repression and the development of arteries in regenerated fins. In contrast, Apela enhanced subintestinal venous plexus formation (SIVP). The use of sunitinib completely inhibited vascular plexus formation in zebrafish, which was not prevented by exogenous application. Furthermore, Apela regulates the expression of vessel remolding-related genes including VWF, IGFPB3, ESM1, VEGFR2, Apln, and Aplnr, thereby linking Apela to the vascular plexus factor network as generated by the STRING online database. Together, our findings reveal a new role for Apela in vessel regeneration and remodeling in fin zebrafish and provide a framework for further understanding the cellular and molecular mechanisms involved in vessel regeneration.
Topics: Animals; Animal Fins; Apelin Receptors; Mammals; Peptide Hormones; Regeneration; Vascular Remodeling; Zebrafish; Zebrafish Proteins
PubMed: 38355946
DOI: 10.1038/s41598-023-50677-1 -
Journal of Medicinal Chemistry Feb 2024The need for anticancer therapies that overcome metallodrug resistance while minimizing adverse toxicities is targeted, herein, using titanium coordination complexes....
The need for anticancer therapies that overcome metallodrug resistance while minimizing adverse toxicities is targeted, herein, using titanium coordination complexes. Octahedral titanium(IV) ,-[Ti{RN(CH-2-MeO-4-R-CH)}] [R = Et, allyl, -Pr, CHO, F, CH(morpholino), the latter from the formyl derivative; R = Me, Et; not all combinations] are attained from Mannich reactions of commercial 2-methoxyphenols (27-74% overall yield, 2 steps). These crystalline (four X-ray structures) Ti(IV)-complexes are active against MCF-7, HCT-116, HT-29, PANC-1, and MDA-MB-468 cancer cell lines (GI = 0.5-38 μM). Their activity and cancer selectivity (vs nontumor MRC-5 cells) typically exceeds that of cisplatin (up to 16-fold). Proteomic analysis (in MCF-7) supported by other studies (G2/M cell cycle arrest, ROS generation, γH2AX production, caspase activation, annexin positivity, western blot, and kinase screens in MCF-7 and HCT-116) suggest apoptosis elicited by more than one mechanism of action. Comparison of these data to the modes of action proposed for salan Ti(IV) complexes is made.
Topics: Humans; Titanium; Amines; Proteomics; Cell Line, Tumor; Antineoplastic Agents; Apoptosis
PubMed: 38331433
DOI: 10.1021/acs.jmedchem.3c01874 -
Frontiers in Cellular Neuroscience 2023High throughput sequencing has generated an enormous amount of information about the genes expressed in various cell types and tissues throughout the body, and about how...
High throughput sequencing has generated an enormous amount of information about the genes expressed in various cell types and tissues throughout the body, and about how gene expression changes over time and in diseased conditions. This knowledge has made targeted gene knockdowns an important tool in screening and identifying the roles of genes that are differentially expressed among specific cells of interest. While many approaches are available and optimized in mammalian models, there are still several limitations in the zebrafish model. In this article, we describe two approaches to target specific genes in the retina for knockdown: cell-penetrating, translation-blocking Vivo-Morpholino oligonucleotides and commercially available lipid nanoparticle reagents to deliver siRNA. We targeted expression of the PCNA gene in the retina of a P23H rhodopsin transgenic zebrafish model, in which rapidly proliferating progenitor cells replace degenerated rod photoreceptors. Retinas collected 48 h after intravitreal injections in adult zebrafish reveal that both Vivo-Morpholinos and lipid encapsulated siRNAs were able to successfully knock down expression of PCNA. However, only retinas injected with Vivo-Morpholinos showed a significant decrease in the formation of P23H rhodopsin-expressing rods, a downstream effect of PCNA inhibition. Surprisingly, Vivo-Morpholinos were able to exit the injected eye and enter the contralateral non-injected eye to inhibit PCNA expression. In this article we describe the techniques, concentrations, and considerations we found necessary to successfully target and inhibit genes through Vivo-Morpholinos and lipid encapsulated siRNAs.
PubMed: 38322239
DOI: 10.3389/fncel.2023.1321337 -
Neurospine Feb 2024To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation.
OBJECTIVE
To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation.
METHODS
Two antisense morpholinos were designed targeting zebrafish best1 to block translation (ATG-MO) or to disrupt splicing (I3E4-MO). Morpholinos were microinjected into fertilized one-cell embryos. Efficacy of splicing morpholino was confirmed by RT-PCR. Phenotypes were analyzed and quantified by microscopy at multiple developmental stages. Neuronal outgrowth was assessed in transgenic zebrafish expressing GFP in neurons. Skeletal ossification was visualized by calcein staining.
RESULTS
Knockdown of best1 resulted in zebrafish embryos with shorter body length, curved axis, low survival rate, microcephaly, reduced eye size, smaller head and brain, impaired neuronal outgrowth, and reduced ossification of craniofacial and vertebral bone.
CONCLUSION
Best1 gene plays critical roles in ophthalmologic, neurological and skeletal development in zebrafish. A patient with a premature stop codon in BEST1 gene exhibited simillar phenotypes, implying a subtype of CVJ malformation.
PubMed: 38317543
DOI: 10.14245/ns.2347238.619