Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Comparative Study of Single-stranded Oligonucleotides Secondary Structure Prediction Tools.

BACKGROUND: Single-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure. RESULTS: The minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with [Formula: see text] of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only [Formula: see text] of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5-10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions. CONCLUSIONS: In general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.

Highly-Specific Single-Stranded Oligonucleotides and Functional Nanoprobes for Clinical Determination of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections.

Early detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) is the key to controlling the spread of these bacterial infections. An important step in developing biosensors involves identifying reliable sensing probes against specific genetic targets for CT and NG. Here, the authors have designed single-stranded oligonucleotides (ssDNAs) targeting mutually conserved genetic regions of cryptic plasmid and chromosomal DNA of both CT and NG. The 5'- and 3'- ends of these ssDNAs are differentially functionalized with thiol groups and coupled with gold nanoparticles (AuNP) to develop absorbance-based assay. The AuNPs agglomerate selectively in the presence of its target DNA sequence and demonstrate a change in their surface plasmon resonance. The optimized assay is then used to detect both CT and NG DNA extracted from 60 anonymized clinical samples with a clinical sensitivity of ∼100%. The limit of detection of the assays are found to be 7 and 5 copies/µL for CT and NG respectively. Furthermore, it can successfully detect the DNA levels of these two bacteria without the need for DNA extraction and via a lateral flow-based platform. These assays thus hold the potential to be employed in clinics for rapid and efficient monitoring of sexually transmitted infections.

Molecular Targets of Aptamers in Gastrointestinal Cancers: Cancer Detection, Therapeutic Applications, and Associated Mechanisms.

Gastrointestinal (GI) cancers are among the most common cancers that impact the global population, with high mortality and low survival rates after breast and lung cancers. Identifying useful molecular targets in GI cancers are crucial for improving diagnosis, prognosis, and treatment outcomes, however, limited by poor targeting and drug delivery system. Aptamers are often utilized in the field of biomarkers identification, targeting, and as a drug/inhibitor delivery cargo. Their natural and chemically modifiable binding capability, high affinity, and specificity are favored over antibodies and potential early diagnostic imaging and drug delivery applications. Studies have demonstrated the use of different aptamers as drug delivery agents and early molecular diagnostic and detection probes for treating cancers. This review aims to first describe aptamers' generation, characteristics, and classifications, also providing insights into their recent applications in the diagnosis and medical imaging, prognosis, and anticancer drug delivery system of GI cancers. Besides, it mainly discussed the relevant molecular targets and associated molecular mechanisms involved, as well as their applications for potential treatments for GI cancers. In addition, the current applications of aptamers in a clinical setting to treat GI cancers are deciphered. In conclusion, aptamers are multifunctional molecules that could be effectively used as an anticancer agent or drug delivery system for treating GI cancers and deserve further investigations for clinical applications.

Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system is a technology that is used to perform site-specific gene disruption, repair, and the modification of genomic DNA via DNA repair mechanisms, and is expected to be a fundamental therapeutic strategy for the treatment of infectious diseases and genetic disorders. For clinical applications, the non-viral vector-based delivery of the CRISPR/Cas ribonucleoprotein (RNP) is important, but the poor efficiency of delivery and the lack of a practical method for its manufacture remains as an issue. We report herein on the development of a lipid nanoparticle (LNP)-based Cas RNP delivery system based on optimally designed single stranded oligonucleotides (ssODNs) that allow efficient in vivo genome editing. The formation of sequence-specific RNP-ssODN complexes was found to be important for the functional delivery of RNP. Furthermore, the melting temperature (Tm) between sgRNA and ssODN had a significant effect on in vivo gene knockout efficiency. An ssODN with a high Tm resulted in limited knockout (KO) activity while that at near room temperature showed the highest KO activity, indicating the importance of the cytosolic release of RNPs. Two consecutive intravenous injections of the Tm optimized formulation achieved approximately 70% and 80% transthyretin KO at the DNA and protein level, respectively, without any obvious toxicity. These findings represent a significant contribution to the development of safe in vivo CRISPR/Cas RNP delivery technology and its practical application in genome editing therapies.

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON's antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide.

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.

A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection.

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning.

BACKGROUND: Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity. RESULTS: A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5' and 3' ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system. CONCLUSIONS: These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates.

SIDT2 mediates gymnosis, the uptake of naked single-stranded oligonucleotides into living cells.

Single-stranded oligonucleotides (ssOligos) are efficiently taken up by living cells without the use of transfection reagents. This phenomenon called 'gymnosis' enables the sequence-specific silencing of target genes in various types of cells. Several antisense ssOligos are used for the treatment of human diseases. However, the molecular mechanism underlying the uptake of naked ssOligos into cells remains to be elucidated. Here, we show that systemic RNA interference deficient-1 (SID-1) transmembrane family 2 (SIDT2), a mammalian ortholog of the Caenorhabditis elegans double-stranded RNA channel SID-1, mediates gymnosis. We show that the uptake of naked ssOligos into cells is significantly downregulated by knockdown of SIDT2. Furthermore, knockdown of SIDT2 inhibited the effect of antisense RNA mediated by gymnosis. Overexpression of SIDT2 enhanced the uptake of naked ssOligos into cells, while a single amino acid mutation in SIDT2 abolished this effect. Our findings highlight the mechanism of extra- and intracellular RNA transport and may contribute to the further development of nucleic acid-based therapies.

Enzymatic Synthesis of Single-Stranded Clonal Pure Oligonucleotides.

Single-stranded oligonucleotides, or oligodeoxyribonucleotides (ODNs), are very important in several fields of science such as molecular biology, diagnostics, nanotechnology, and gene therapy. They are usually chemically synthesized. Here we describe an enzymatic method which enables us to synthesize pure oligonucleotides which can be up to several hundred long bases.

LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.

Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells.