Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing.

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.

Nanoparticles-assisted aptamer biosensing for the detection of environmental pathogens.

Given the importance of public health, it is crucial to develop quick, targeted, highly sensitive, and accurate technologies to monitor pathogenic microbes in response to the growing concerns of food and environmental safety. Although conventional approaches for microbiological detection are available, they are laborious, and often skill demanding. Therefore, such approaches are incompetent in the on-site or high-throughput assessment of pathogenic microbes. Numerous efforts have been made to develop biosensors that use nucleic acid aptamer as the biorecognition element, which would avoid the abovementioned limitations. Incorporating nanomaterials (NMs) into aptamer-based biosensors (aptasensors) improves their sensitivity and specificity, opening exciting possibilities for various applications, such as bioanalysis of food and environmental samples. Over the last decade, nanomaterial-conjugated aptasensors have seen a steadily rising demand. To this end, the main goal of this study is to demonstrate the novelty in the design of nanomaterial-conjugated aptasensors and how they can be used to detect different pathogenic microbes in water and food. The intent of this paper is to evaluate the cutting-edge techniques that have appeared in nano-aptasensors throughout the past few years, such as manufacturing procedures, analytical credibility, and sensing mechanisms. Additionally, the fundamental performance parameters of aptasensing techniques (such as detection limits, and sensing ranges response) were also used to evaluate their practical applicability. Finally, it is anticipated that this study will inspire innovative ideas and techniques for the construction and use of aptasensors for monitoring pathogenic microorganisms in food, drinks, recreational water, and wastewater.

Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2'-O-methyl gapmer oligonucleotides.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes.

2'-O-Methyl (2'-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2'-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2'-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications.

How to Develop and Prove High-Efficiency Selection of Ligands from Oligonucleotide Libraries: A Universal Framework for Aptamers and DNA-Encoded Small-Molecule Ligands.

Screening molecular libraries for ligands capable of binding proteins is widely used for hit identification in the early drug discovery process. Oligonucleotide libraries provide a very high diversity of compounds, while the combination of the polymerase chain reaction and DNA sequencing allow the identification of ligands in low copy numbers selected from such libraries. Ligand selection from oligonucleotide libraries requires mixing the library with the target followed by the physical separation of the ligand-target complexes from the unbound library. Cumulatively, the low abundance of ligands in the library and the low efficiency of available separation methods necessitate multiple consecutive rounds of partitioning. Multiple rounds of inefficient partitioning make the selection process ineffective and prone to failures. There are continuing efforts to develop a separation method capable of reliably generating a pure pool of ligands in a single round of partitioning; however, none of the proposed methods for single-round selection have been universally adopted. Our analysis revealed that the developers' efforts are disconnected from each other and hindered by the lack of quantitative criteria of selection quality assessment. Here, we present a formalism that describes single-round selection mathematically and provides parameters for quantitative characterization of selection quality. We use this formalism to define a universal strategy for development and validation of single-round selection methods. Finally, we analyze the existing partitioning methods, the published single-round selection reports, and some pertinent practical considerations through the prism of this formalism. This formalism is not an experimental protocol but a framework for correct development of experimental protocols. While single-round selection is not a goal by itself and may not always suffice selection of good-quality ligands, our work will help developers of highly efficient selection approaches to consolidate their efforts under an umbrella of universal quantitative criteria of method development and assessment.

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

Oligonucleotides containing 2'-O-methyl-5-(1-phenyl-1,2,3-triazol-4-yl)uridines demonstrate increased affinity for RNA and induce exon-skipping in vitro.

The nucleotide monomer containing the 1-phenyl-1,2,3-triazole group attached to the 5-position of 2'-O-methyluridine is hereby presented together with a derivative further substituted with a p-sulfonamide group on the phenyl ring. Both were conveniently synthesised, and synergistic effect of the modifications were demonstrated when introduced into oligonucleotides and hybridised to complementary RNA. The combination of stacking of the phenyltriazoles and the conformational steering from the 2'-OMe group gave thermally very stable duplexes. Exon skipping in the distrophin transcript using 20-mer 2'-OMePS sequences with two phenyltriazoles introduced in different positions with and without the sulfonamide demonstrated efficient exon skipping but at the same level as the 2'-OMePS reference ASO.

Recent Advances in Oligonucleotide Therapeutics in Oncology.

Cancer is one of the leading causes of death worldwide. Conventional therapies, including surgery, radiation, and chemotherapy have achieved increased survival rates for many types of cancer over the past decades. However, cancer recurrence and/or metastasis to distant organs remain major challenges, resulting in a large, unmet clinical need. Oligonucleotide therapeutics, which include antisense oligonucleotides, small interfering RNAs, and aptamers, show promising clinical outcomes for disease indications such as Duchenne muscular dystrophy, familial amyloid neuropathies, and macular degeneration. While no approved oligonucleotide drug currently exists for any type of cancer, results obtained in preclinical studies and clinical trials are encouraging. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in oncology, review current clinical trials, and discuss associated challenges.

Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro.

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2'-O-methyl phosphorothioate (2'-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2'-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2'-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.

Efficient Epidermal Growth Factor Receptor Targeting Oligonucleotide as a Potential Molecule for Targeted Cancer Therapy.

Epidermal growth factor receptor (EGFR) is associated with the progression of a wide range of cancers including breast, glioma, lung, and liver cancer. The observation that EGFR inhibition can limit the growth of EGFR positive cancers has led to the development of various EGFR inhibitors including monoclonal antibodies and small-molecule inhibitors. However, the reported toxicity and drug resistance greatly compromised the clinical outcome of such inhibitors. As a type of chemical antibodies, nucleic acid aptamer provides an opportunity to overcome the obstacles faced by current EGFR inhibitors. In this study, we have developed and investigated the therapeutic potential of a 27mer aptamer CL-4RNV616 containing 2'-O-Methyl RNA and DNA nucleotides. Our results showed that CL-4RNV616 not only displayed enhanced stability in human serum, but also effectively recognized and inhibited the proliferation of EGFR positive Huh-7 liver cancer, MDA-MB-231 breast cancer, and U87MG glioblastoma cells, with an IC50 value of 258.9 nM, 413.7 nM, and 567.9 nM, respectively. Furthermore, TUNEL apoptosis assay revealed that CL-4RNV616 efficiently induced apoptosis of cancer cells. In addition, clinical breast cancer biopsy-based immunostaining assay demonstrated that CL-4RNV616 had a comparable detection efficacy for EGFR positive breast cancer with commonly used commercial antibodies. Based on the results, we firmly believe that CL-4RNV616 could be useful in the development of targeted cancer therapeutics and diagnostics.

Development of Novel antimiRzymes for Targeted Inhibition of miR-21 Expression in Solid Cancer Cells.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the regulation of gene expression. Previous reports showed an over-expression of miRNA-21 (miR-21) in various cancer cells, and its up-regulation is closely related to cancer initiation, proliferation and metastasis. In this work, we envisioned the development of novel antimiRzymes (anti-miRNA-DNAzyme) that are capable of selectively targeting and cleaving miR-21 and inhibit its expression in cancer cells using the DNAzyme technique. For this purpose, we have designed different antimiRzyme candidates by systematically targeting different regions of miR-21. Our results demonstrated that RNV541, a potential arm-loop-arm type antimiRzyme, was very efficient (90%) to suppress miR-21 expression in U87MG malignant glioblastoma cell line at 200 nM concentration. In addition, RNV541 also inhibited miR-21 expression (50%) in MDA-MB-231 breast cancer cell line. For targeted delivery, we conjugated RNV541 with a transferrin receptor (TfR) targeting aptamer for TfR-mediated cancer cell delivery. As expected, the developed chimeric structure efficiently delivered the antimiRzyme RNV541 into TfR positive glioblastoma cells. TfR aptamer-RNV541 chimeric construct showed 52% inhibition of miR-21 expression in U87MG glioblastoma cells at 2000 nM concentration, without using any transfection reagents, making it a highly desirable strategy to tackle miR-21 over-expressed malignant cancers. Although these are in vitro based observations, based on our results, we firmly believe that our findings could be beneficial towards the development of targeted cancer therapeutics where conventional therapies face several challenges.

Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides.

Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.

Next-Generation Nucleic Acid Aptamers with Two-Base-Modified Nucleotides Have Improved Binding Affinity and Potency.

Twice as apt: Nucleic acid aptamers with high binding affinity, specificity, epitope coverage and nuclease resistance were developed by using libraries containing oligonucleotides in which two bases in the pyrimidine nucleotide had been modified.

Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule.

The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3 ), unlocked nucleic acid (UNA) and 3'-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

Construction of a Bivalent Thrombin Binding Aptamer and Its Antidote with Improved Properties.

Aptamers are short synthetic DNA or RNA oligonucleotides that adopt secondary and tertiary conformations based on Watson-Crick base-pairing interactions and can be used to target a range of different molecules. Two aptamers, HD1 and HD22, that bind to exosites I and II of the human thrombin molecule, respectively, have been extensively studied due to their anticoagulant potentials. However, a fundamental issue preventing the clinical translation of many aptamers is degradation by nucleases and reduced pharmacokinetic properties requiring higher dosing regimens more often. In this study, we have chemically modified the design of previously described thrombin binding aptamers targeting exosites I, HD1, and exosite II, HD22. The individual aptamers were first modified with an inverted deoxythymidine nucleotide, and then constructed bivalent aptamers by connecting the HD1 and HD22 aptamers either through a triethylene glycol (TEG) linkage or four consecutive deoxythymidines together with an inverted deoxythymidine nucleotide at the 3'-end. The anticoagulation potential, the reversal of coagulation with different antidote sequences, and the nuclease stability of the aptamers were then investigated. The results showed that a bivalent aptamer RNV220 containing an inverted deoxythymidine and a TEG linkage chemistry significantly enhanced the anticoagulation properties in blood plasma and nuclease stability compared to the existing aptamer designs. Furthermore, a bivalent antidote sequence RNV220AD efficiently reversed the anticoagulation effect of RNV220 in blood plasma. Based on our results, we believe that RNV220 could be developed as a potential anticoagulant therapeutic molecule.

Development of Cell-Specific Aptamers: Recent Advances and Insight into the Selection Procedures.

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA/2'-O-Methyl Mixmer Antisense Oligonucleotide.

In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2'-O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2'-OMePS) to the corresponding fully modified 2'-O-methyl RNA AO (2'-OMePS) as a control. Our results showed that the MNA/2'-OMePS efficiently induced exon 23 skipping. As expected, the 2'-OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides.

Smart functional nucleic acid chimeras: enabling tissue specific RNA targeting therapy.

A major obstacle for effective utilization of therapeutic oligonucleotides such as siRNA, antisense, antimiRs etc. is to deliver them specifically to the target tissues. Toward this goal, nucleic acid aptamers are re-emerging as a prominent class of biomolecules capable of delivering target specific therapy and therapeutic monitoring by various molecular imaging modalities. This class of short oligonucleotide ligands with high affinity and specificity are selected from a large nucleic acid pool against a molecular target of choice. Poor cellular uptake of therapeutic oligonucleotides impedes gene-targeting efficacy in vitro and in vivo. In contrast, aptamer-oligonucleotide chimeras have shown the capacity to deliver siRNA, antimiRs, small molecule drugs etc. toward various targets and showed very promising results in various studies on different diseases models. However, to further improve the bio-stability of such chimeric conjugates, it is important to introduce chemically-modified nucleic acid analogs. In this review, we highlight the applications of nucleic acid aptamers for target specific delivery of therapeutic oligonucleotides.

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold.

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.