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Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2’-O-methyl and 2’-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5−20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.

Antibiotic administration is the standard treatment for the bacterium Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer. However, the long-term consequences of this treatment on the human indigenous microbiota are relatively unexplored. Here we studied short- and long-term effects of clarithromycin and metronidazole treatment, a commonly used therapy regimen against H. pylori, on the indigenous microbiota in the throat and in the lower intestine. The bacterial compositions in samples collected over a four-year period were monitored by analyzing the 16S rRNA gene using 454-based pyrosequencing and terminal-restriction fragment length polymorphism (T-RFLP). While the microbial communities of untreated control subjects were relatively stable over time, dramatic shifts were observed one week after antibiotic treatment with reduced bacterial diversity in all treated subjects in both locations. While the microbiota of the different subjects responded uniquely to the antibiotic treatment some general trends could be observed; such as a dramatic decline in Actinobacteria in both throat and feces immediately after treatment. Although the diversity of the microbiota subsequently recovered to resemble the pre treatment states, the microbiota remained perturbed in some cases for up to four years post treatment. In addition, four years after treatment high levels of the macrolide resistance gene erm(B) were found, indicating that antibiotic resistance, once selected for, can persist for longer periods of time than previously recognized. This highlights the importance of a restrictive antibiotic usage in order to prevent subsequent treatment failure and potential spread of antibiotic resistance.

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5′ untranslated region (5′ UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5′ UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications.

Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3' ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred .

Gapmer antisense oligonucleotides cleave target RNA effectively in vivo , and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.

Conditionally activated (“caged”) oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical.

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

We report the development of on-chip fluorescence switching system based on DNA strand displacement and DNA hybridization for the construction of a rewritable and randomly accessible data storage device. In this study, the feasibility and potential effectiveness of our proposed system was evaluated with a series of wet experiments involving 40 bits (5 bytes) of data encoding a 5-charactered text (KRIBB). Also, a flexible data rewriting function was achieved by converting fluorescence signals between “ON” and “OFF” through DNA strand displacement and hybridization events. In addition, the proposed system was successfully validated on a microfluidic chip which could further facilitate the encoding and decoding process of data. To the best of our knowledge, this is the first report on the use of DNA hybridization and DNA strand displacement in the field of data storage devices. Taken together, our results demonstrated that DNA-based fluorescence switching could be applicable to construct a rewritable and randomly accessible data storage device through controllable DNA manipulations.

Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown . Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.