Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

• MeSH
• Agrobacterium tumefaciens genetics   metabolism   MeSH
• RNA, Antisense biosynthesis   genetics   metabolism   MeSH
• RNA, Double-Stranded analysis   chemistry   metabolism   MeSH
• Transcription, Genetic MeSH
• Plants, Genetically Modified genetics   MeSH
• Molecular Sequence Data MeSH
• Antibodies, Monoclonal immunology   MeSH
• RNA, Viral antagonists & inhibitors   metabolism   MeSH
• Base Sequence MeSH
• Solanum tuberosum genetics   virology   MeSH
• Transformation, Genetic MeSH
• Viroids genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Antisense MeSH
• RNA, Double-Stranded MeSH
• Antibodies, Monoclonal MeSH
• RNA, Viral MeSH

The first observation of chromosomally encoded small antisense RNA in Corynebacterium glutamicum is reported. Transcription oriented in the reverse direction to the transcription of the genes cg1934 and cg1935 was demonstrated within the chromosomal cg1934-cg1935 intergenic region. The transcription was found to be increased after heat shock. The transcriptional start point of this RNA designated ArnA was localized 21 bp upstream of the cg1935 translational start point by primer extension analysis, when the total RNA was isolated from cells grown at 30 degrees C. After heat shock, the transcriptional start point of an additional species of ArnA RNA was detected 19 bp upstream of the cg1935 translational start point. The stress-response sigma factor SigH was found to be involved in the synthesis of ArnA RNAs. The 3' end of the ArnA RNAs was identified using the 3'-rapid amplification of cDNA ends technique. The length of the two ArnA RNA species was thus determined to be 129 and 131 nt, respectively. The ArnA RNAs were found to overlap the 5'-untranslated region of the transcript of the cg1935 gene coding for a transcriptional regulator of the GntR family. These results suggest that the noncoding ArnA RNAs have a regulatory function.

• MeSH
• 5' Untranslated Regions genetics   MeSH
• RNA, Antisense genetics   MeSH
• Chromosomes, Bacterial MeSH
• Genes, Bacterial MeSH
• Bacterial Proteins metabolism   MeSH
• RNA, Bacterial genetics   MeSH
• Corynebacterium glutamicum genetics   MeSH
• Transcription, Genetic MeSH
• DNA, Intergenic MeSH
• Nucleic Acid Conformation MeSH
• Molecular Sequence Data MeSH
• Transcription Initiation Site MeSH
• Gene Expression Regulation, Bacterial MeSH
• Base Sequence MeSH
• Sigma Factor metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5' Untranslated Regions MeSH
• RNA, Antisense MeSH
• Bacterial Proteins MeSH
• RNA, Bacterial MeSH
• DNA, Intergenic MeSH
• SigH protein, bacteria MeSH Browser
• Sigma Factor MeSH

Insertion sequences (IS) represent mobile genetic elements that have been shown to be associated with bacterial evolution and adaptation due to their effects on genome plasticity. In Bordetella pertussis, the causative agent of whooping cough, the numerous IS elements induce genomic rearrangements and contribute to the diversity of the global B. pertussis population. Previously, we have shown that the majority of IS-specific endogenous promoters induce the synthesis of alternative transcripts and thereby affect the transcriptional landscape of B. pertussis. Here, we describe the regulatory RNA Rfi2, which is transcribed from the Pout promoter of the IS481 gene BP1118 antisense to the adjacent fim2 gene encoding the major serotype 2 fimbrial subunit of B. pertussis. Among the classical bordetellae, Rfi2 is unique to B. pertussis, suggesting its specific role in virulence. We show that Rfi2 RNA attenuates fim2 transcription and, consequently, the production of the Fim2 protein. Interestingly, the mutant that does not produce Rfi2 displayed significantly increased cytotoxicity towards human macrophages compared to the parental strain. This observation suggests that the Rfi2-mediated reduction in cytotoxicity represents an evolutionary adaptation of B. pertussis that fine-tunes its interaction with the human host. Given the immunogenicity of Fim2, we further hypothesize that Rfi2-mediated modulation of Fim2 production contributes to immune evasion. To our knowledge, Rfi2 represents the first functionally characterized IS element-driven antisense RNA that modulates the expression of a virulence gene.

• Keywords
• Bordetella pertussis, antisense RNA, cytotoxicity towards macrophages, fimbriae serotype 2, insertion sequence, modulation of virulence,
• MeSH
• RNA, Antisense * genetics   metabolism   MeSH
• Fimbriae, Bacterial * genetics   metabolism   MeSH
• Bordetella pertussis * genetics   pathogenicity   metabolism   MeSH
• Virulence Factors, Bordetella genetics   MeSH
• Humans MeSH
• Macrophages microbiology   MeSH
• Whooping Cough microbiology   MeSH
• Promoter Regions, Genetic MeSH
• Fimbriae Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Serogroup MeSH
• DNA Transposable Elements * MeSH
• Virulence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Bacterial MeSH
• RNA, Antisense * MeSH
• Virulence Factors, Bordetella MeSH
• fim2 protein, Bordetella MeSH Browser
• Fimbriae Proteins * MeSH
• DNA Transposable Elements * MeSH

The effect of oligodeoxyribonucleotides complementary to the region of the so-called pathogenicity domain (nucleotides 42-78) of the upper RNA strand of potato spindle tuber viroid (PSTVd) (severe) on viroid infection was investigated. The oligonucleotides were allowed to form hybrids with PSTVd in the infection mixtures before inoculation. Infectivity tests were performed using intact plants and plant protoplasts. It was found that the DNA oligonucleotides caused significant reduction of viroid infection at plant and single cell levels. The 200-fold molar excess of antisense DNA over viroid RNA is usually sufficient for the complete blocking of viroid infection. The inhibitory effect is strongly sequence specific. Inhibition by corresponding antisense RNA was much less efficient than that caused by antisense DNA.

• MeSH
• DNA, Antisense pharmacology   MeSH
• Molecular Sequence Data MeSH
• Plant Diseases microbiology   MeSH
• Oligonucleotides MeSH
• Base Sequence MeSH
• Solanum tuberosum microbiology   MeSH
• Viroids drug effects   genetics   MeSH
• Virus Diseases prevention & control   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Antisense MeSH
• Oligonucleotides MeSH

Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.

• MeSH
• Oligonucleotides, Antisense * therapeutic use   genetics   MeSH
• Genetic Diseases, Inborn genetics   therapy   MeSH
• Humans MeSH
• Morpholinos therapeutic use   genetics   MeSH
• Mutation * MeSH
• RNA Splicing * MeSH
• Rare Diseases * genetics   drug therapy   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Oligonucleotides, Antisense * MeSH
• Morpholinos MeSH

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

• Keywords
• Fuchs endothelial corneal dystrophy, RNA toxicity, antisense oligonucleotide, corneal dystrophy, non-coding mutations, repeat-expansion, transcription factor 4, triplet repeat-mediated disease,
• MeSH
• Oligonucleotides, Antisense pharmacology   MeSH
• Cell Nucleus drug effects   metabolism   MeSH
• Endothelial Cells metabolism   MeSH
• Trinucleotide Repeat Expansion genetics   MeSH
• Fuchs' Endothelial Dystrophy genetics   pathology   MeSH
• Genetic Predisposition to Disease * MeSH
• Cohort Studies MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Organ Specificity MeSH
• RNA Processing, Post-Transcriptional MeSH
• RNA Precursors genetics   MeSH
• Risk Factors MeSH
• Endothelium, Corneal pathology   MeSH
• Aged MeSH
• RNA Splicing Factors metabolism   MeSH
• Transcription Factor 4 genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oligonucleotides, Antisense MeSH
• RNA, Messenger MeSH
• RNA Precursors MeSH
• RNA Splicing Factors MeSH
• TCF4 protein, human MeSH Browser
• Transcription Factor 4 MeSH

BACKGROUND: The genetic and epigenetic alterations observed in acute myeloid leukemia (AML) contribute to its heterogeneity, influencing disease progression response to therapy, and patient outcomes. The use of antisense oligonucleotides (ASOs) technology allows for the design of oligonucleotide inhibitors based on gene sequence information alone, enabling precise targeting of key molecular pathways or specific genes implicated in AML. METHODS AND RESULTS: Midostaurin, a FLT3 specific inhibitor and ASOs targeting particular genes, exons, or mutations was conducted using AML models. This ASOs treatment was designed to bind to exon 7 of the MBNL1 (muscleblind-like) gene. Another target was the FLT3 gene, focusing on two aspects: (a) FLT3-ITD (internal tandem duplication), to inhibit the expression of this aberrant gene form, and (b) the FLT3 in general. Treated and untreated cells were analyzed using quantitative PCR (qPCR), dot blot, and Raman spectroscopy. This study contrasts midostaurin with ASOs that inhibit FLT3 protein production or its isoforms via mRNA degradation. A trend of increased FLT3 expression was observed in midostaurin-treated cells, while ASO-treated cells showed decreased expression, though these changes were not statistically significant. CONCLUSIONS: In AML, exon 7 of MBNL1 is involved in several cellular processes and in this study, exon 7 of MBNL1 was targeted for method optimization, with the highest block of the exon 7 gene variant observed 48 h post-transfection. Midostaurin, a multitargeted kinase inhibitor, acts against the receptor tyrosine kinase FLT3, a critical molecule in AML pathogenesis. While midostaurin blocks FLT3 signaling pathways, it paradoxically increases FLT3 expression.

• Keywords
• Acute myeloid leukemia, Antisense oligonucleotides, FLT3, MBNL1, Target specific therapy,
• MeSH
• Leukemia, Myeloid, Acute * genetics   drug therapy   MeSH
• Oligonucleotides, Antisense * pharmacology   genetics   MeSH
• Exons genetics   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Leukemic drug effects   MeSH
• Staurosporine * analogs & derivatives   pharmacology   MeSH
• fms-Like Tyrosine Kinase 3 * genetics   antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Oligonucleotides, Antisense * MeSH
• FLT3 protein, human MeSH Browser
• MBNL1 protein, human MeSH Browser
• midostaurin MeSH Browser
• RNA-Binding Proteins MeSH
• Staurosporine * MeSH
• fms-Like Tyrosine Kinase 3 * MeSH

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

• Keywords
• Antisense morpholino oligonucleotide, Dead end gene, Germ line chimera, Primordial germ cell, Sterilization, Sturgeon,
• MeSH
• Oligonucleotides, Antisense * administration & dosage   MeSH
• Cell Death MeSH
• Gene Knockdown Techniques methods   veterinary   MeSH
• Gonads chemistry   MeSH
• DNA, Complementary chemistry   MeSH
• Morpholinos * administration & dosage   MeSH
• RNA-Binding Proteins analysis   genetics   MeSH
• Fishes * genetics   MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Sterilization, Reproductive methods   veterinary   MeSH
• Germ Cells physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oligonucleotides, Antisense * MeSH
• DNA, Complementary MeSH
• Morpholinos * MeSH
• RNA-Binding Proteins MeSH

We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.

• MeSH
• Oligonucleotides, Antisense pharmacology   MeSH
• Cell Line MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Hepatitis B Surface Antigens genetics   metabolism   MeSH
• Carcinoma, Hepatocellular microbiology   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Molecular Sequence Data MeSH
• Gene Expression Regulation, Viral drug effects   MeSH
• Base Sequence MeSH
• Genes, Viral drug effects   MeSH
• Viral Structural Proteins genetics   MeSH
• Hepatitis B virus genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Oligonucleotides, Antisense MeSH
• Hepatitis B Surface Antigens MeSH
• RNA, Messenger MeSH
• Viral Structural Proteins MeSH

Purine and pyrimidine 4'-alkoxy-2'-deoxynucleosides were efficiently prepared from nucleoside 4'-5'-enol acetates in three steps by N-iodosuccinimide promoted alkoxylation, hydrolysis, and reduction followed by conversion to phosphoramidite monomers for the solid-phase synthesis of the oligonucleotides. Fully modified 4'-alkoxyoligodeoxynucleotides, which are characterized by a prevalent N-type (RNA-like) conformation, exhibited superior chemical and nuclease resistance as well as excellent hybridization properties with a strong tendency for RNA-selective hybridization, suggesting a potential application of 4'-alkoxy-oligodeoxynucleotides in antisense technologies.

• MeSH
• Oligonucleotides, Antisense chemistry   MeSH
• Nucleic Acid Hybridization MeSH
• Nucleic Acid Conformation MeSH
• Molecular Conformation MeSH
• Molecular Structure MeSH
• Oligodeoxyribonucleotides chemical synthesis   chemistry   MeSH
• Purines chemistry   MeSH
• RNA chemistry   MeSH
• Succinimides chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oligonucleotides, Antisense MeSH
• N-iodosuccinimide MeSH Browser
• Oligodeoxyribonucleotides MeSH
• purine MeSH Browser
• Purines MeSH
• RNA MeSH
• Succinimides MeSH