A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides.

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.

Dual-modified antisense oligonucleotides targeting oncogenic protocadherin to treat gastric cancer.

BACKGROUND: The objective of this study was to develop an innovative treatment strategy utilizing antisense oligonucleotides (ASOs) that target the gene encoding protocadherin alpha 11 (PCDHA11) and to elucidate the role of PCDHA11 in gastric cancer cells. METHODS: We designed and screened 54 amido-bridged nucleic acid (AmNA)-modified ASOs, selecting them based on PCDHA11-knockdown efficacy, in vitro and in vivo activity, and off-target effects. We assessed the impact of AmNA-modified anti-PCDHA11 ASOs on cellular functions and signaling pathways, and investigated the effects of Pcdha11 deficiency in mice. RESULTS: AmNA-modified anti-PCDHA11 ASOs significantly reduced the proliferation of gastric cancer cells and other solid tumors, whereas overexpression of PCDHA11 enhanced cell proliferation. The selected ASOs inhibited cellular functions related to the metastatic potential of gastric cancer cells, including migration, invasiveness, spheroid formation, and cancer stemness. Our findings revealed that AmNA-modified anti-PCDHA11 ASOs disrupted the AKT/mTOR, Wnt/ß-catenin, and JAK/STAT signaling pathways. In mouse models of peritoneal metastasis (gastric and pancreatic cancer), systemic metastasis, and established subcutaneous tumors, administration of AmNA-modified anti-PCDHA11 ASOs inhibited tumor growth. ASO treatment induced reversible, dose- and sequence-dependent liver damage. Pcdha11-deficient mice demonstrated normal reproductive, organ, and motor functions. CONCLUSIONS: AmNA-modified anti-PCDHA11 ASOs offer a promising therapeutic strategy for the treatment of gastric cancer and other solid malignancies.

Lantern-type G-quadruplex fluorescent sensors for detecting divalent metal ions.

Three thioflavin T (ThT) derivatives, namely ThT/ethylenediaminetetraacetic acid conjugates (E1T, E2T, and E1T1P), were designed and synthesized as sensing components for divalent metal ion detection. Furthermore, these ThT derivatives were used to design lantern-type G-quadruplex (G4) fluorescent sensors. The fluorescence intensities of the ThT derivatives decreased by 1.2- to 5.6-folds in the presence of Ni2+ and Cu2+, respectively, regardless of the topology of the utilized G4. Conversely, when Mn2+ and Zn2+ coexisted in antiparallel G4, the fluorescence intensities of E2T increased to approximately 3.3- and 2.3-folds, respectively, depending on the concentration of the divalent metal ion, allowing for quantitative analyses. The Job plot analysis revealed that the binding ratio of G4 and E2T changed from 2:1 to 1:2 with the increasing concentration of the divalent metal ions. These results indicated that the basic principle of such a lantern-type G4 sensor can be applied to the detection of divalent metal ions and other types of targets, such as proteins, and small molecules via ThT derivatization.

Preclinical toxicological assessment of amido-bridged nucleic acid-modified antisense oligonucleotides targeting synaptotagmin XIII for intra-abdominal treatment of peritoneal metastasis of gastric cancer.

BACKGROUND: Peritoneal metastasis of gastric cancer is closely associated with dismal prognosis. In previous preclinical proof-of-concept studies, an amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotide (ASO), designated ASO-4733 that targets the gene encoding synaptotagmin XIII (SYT13), inhibited cellular functions required for the formation of peritoneal metastasis of gastric cancer cells. ASO-4733 achieved therapeutic effects when intra-abdominally administered to mouse xenograft models. Here, we conducted an analysis of Syt13-deficient mice to determine the pharmacokinetics and toxicity of intra-abdominal administration of ASO-4733. METHODS: The effects of Syt13-deficiency in mice were determined. Good Laboratory Practice toxicity tests and the toxicokinetics of intra-abdominal administration of ASO-4733 were conducted in cynomolgus monkeys and rats. The pharmacokinetics of ASO-4733 administered intravenously or intra-abdominally to rats were investigated. RESULTS: Syt13-deficient mice exhibited normal reproduction, organ functions, and motor functions. Weekly intra-abdominal administration of ASO-4733 (125 mg/kg), corresponding to a 50-fold increase of the estimated clinical dose for 4 weeks, was well tolerated by cynomolgus monkeys. In rats, off-target toxicity (not attributable to hybridization) was observed after weekly intra-abdominal administration of ASO-4733. Blood concentrations of ASO-4733 were lower and rose more slowly after intra-abdominal administration compared with intravenous administration. CONCLUSIONS: The preclinical profile of intra-abdominal administration of ASO-4733 demonstrated its suitability for entry into clinical trials of patients with peritoneal metastasis of gastric cancer.

In vitro screening of chemically synthesized dipeptide-antisense oligonucleotide conjugates to identify ligand molecules enhancing their activity.

Oligonucleotide therapeutics, particularly antisense oligonucleotides (ASOs), have emerged as promising candidates in drug discovery. However, their effective delivery to the target tissues and cells remains a challenge, necessitating the development of suitable drug delivery technologies for ASOs to enable their practical application. In this study, we synthesized a library of chemically modified dipeptide-ASO conjugates using a recent synthetic method based on the Ugi reaction. We then conducted in vitro screening of this library using luciferase-expressing cell lines to identify ligands capable of enhancing ASO activity. Our findings suggest that N-(4-nitrophenoxycarbonyl)glycine may interact with the thiophosphate moiety of the phosphorothioate-modification in ASO. Through our screening efforts, we identified two ligands that modestly reduced luciferase luminescence in a cell type-selective manner. Furthermore, quantification of luciferase mRNA levels revealed that one of these promising dipeptide-ASO conjugates markedly suppressed luciferase RNA levels through its antisense effect in prostate-derived DU-145 cells compared to the ASOs without ligand modification.

Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides.

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.

Synthesis and biophysical properties of tetravalent PEG-conjugated antisense oligonucleotide.

This study was aimed at developing a novel platform for tetravalent conjugation of 4-arm polyethylene glycol (PEG) with an antisense oligonucleotide (ASO). The ASO technology has several limitations, such as low cellular uptake, poor nuclease stability, and short half-life. PEG-conjugated ASOs may result in an improvement in the pharmacokinetic behavior of the drug. Moreover, PEGylation can reduce enzymatic degradation and renal excretion of the conjugates, thereby, increasing its blood stability and retention time. In this study, we successfully synthesized PEG-ASO conjugate consisting of 4-arm-PEG and four molecules of ASO (4-arm-PEG-tetra ASO). Its hybridization ability with complementary RNA, enzymatic stability, and in vitro gene silencing ability were evaluated. No significant difference in hybridization ability was observed between 4-arm-PEG-tetra ASO and the parent ASO. In addition, gene silencing activity of the 4-arm-PEG-tetra ASO was observed in vitro. However, the in vitro activity of the 4-arm-PEG-tetra ASO was slightly reduced as that of the parent ASO. Moreover, the 4-arm-PEG-tetra ASO showed appreciable stability in cellular extract, suggesting that it hybridizes with mRNA in its intact form, without being cleaved in the cell, and exhibits ASO activity.

Systematic Analysis of 2'-O-Alkyl Modified Analogs for Enzymatic Synthesis and Their Oligonucleotide Properties.

Enzymatic oligonucleotide synthesis is used for the development of functional oligonucleotides selected by in vitro selection. Expanding available sugar modifications for in vitro selection helps the functional oligonucleotides to be used as therapeutics reagents. We previously developed a KOD DNA polymerase mutant, KOD DGLNK, that enzymatically synthesized fully-LNA- or 2'-O-methyl-modified oligonucleotides. Here, we report a further expansion of the available 2'-O-alkyl-modified nucleotide for enzymatic synthesis by KOD DGLNK. We chemically synthesized five 2'-O-alkyl-5-methyluridine triphosphates and incorporated them into the oligonucleotides. We also enzymatically synthesized a 2'-O-alkyl-modified oligonucleotide with a random region (oligonucleotide libraries). The 2'-O-alkyl-modified oligonucleotide libraries showed high nuclease resistance and a wide range of hydrophobicity. Our synthesized 2'-O-alkyl-modified oligonucleotide libraries provide novel possibilities that can promote the development of functional molecules for therapeutic use.

Azobenzene-modified DNA aptamers evolved by capillary electrophoresis (CE)-SELEX method.

Chemically modified aptamers have recently emerged as important materials for nucleic acid based therapeutics and diagnostic tools. Here, we report in vitro evolution of azobenzene-modified DNA aptamers by capillary electrophoresis (CE)-SELEX method. Azobenzene has been considered to be a fascinating functional group due to its trans-cis photo-isomerization property. We harnessed C5-azobenzene-modified 2'-deoxyuridine (dUAz) as a azobenzene-tethered unit and subjected it to CE-SELEX with human thrombin. The obtained dUAz-modified aptamer showed strong binding affinity toward human thrombin and could be reversibly photo-isomerized by different wavelengths of light. This work demonstrates that CE-SELEX is a powerful method to obtain chemically modified aptamers and dUAz is an excellent photo-responsive nucleoside for nucleic acid photo-switches.

DNA Polymerase Variants with High Processivity and Accuracy for Encoding and Decoding Locked Nucleic Acid Sequences.

Xenobiotic nucleic acids (XNAs) are chemically modified nucleic acid analogues with potential applications in nucleic acid-based therapeutics including nucleic acid aptamers, ribozymes, small interfering RNAs, and antisense oligonucleotides. We have developed a promising XNA for therapeutic uses, 2',4'-bridged nucleic acid (2',4'-BNA), also known as locked nucleic acid (LNA). Unlike the rational design of small interfering and antisense oligonucleotides, the development of LNA aptamers and catalysts requires genetically engineered polymerases that enable the synthesis of LNA from DNA and the converse reverse transcription. However, no LNA decoders or encoders with sufficient performance have been developed. In this study, we developed variants of KOD DNA polymerase, a family B DNA polymerase derived from Thermococcus kodakarensis KOD1, which are effective LNA decoders and encoders, via structural analyses. KOD DGLNK (KOD: N210D/Y409G/A485L/D614N/E664K) enabled LNA synthesis from DNA (DNA → LNA), and KOD DLK (KOD: N210D/A485L/E664K) enabled LNA reverse transcription to DNA (LNA → DNA). Both variants exhibited greatly improved efficiency and accuracy. Notably, we synthesized LNAs longer than one kilobase using KOD DGLNK. We also showed that these variants can accept 2'-O-methyl (2'-OMe), a common modification for therapeutic uses. Here, we also show that LNA and 2'-OMe mix aptamer can be practically obtained via SELEX. The variants can be used as powerful tools for creating XNA aptamers and catalysts to completely eliminate the natural species, DNA and RNA.

Discovery of cell-internalizing artificial nucleic acid aptamers for lung fibroblasts and targeted drug delivery.

Lung fibroblasts play major roles in the lung repair/fibrosis process through synthesis and remodeling of extracellular matrix. Those aberrant activations and elevated proliferations are associated with several fibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Targeting fibroblasts is a promising approach for preventing aberrant remodeling of lung architecture and protect irreversible pulmonary fibrosis. In this study, we developed an aptamer that can target lung fibroblasts and explored its potential as a delivery vehicle of cytotoxic agents intracellularly. The aptamer was discovered from artificial nucleic acid libraries through cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). This indole-modified aptamer can bind to LL97A cells, a fibroblast cell line derived from IPF patients, with high affinity (Kd = 70 nM). It also showed affinity to other lung fibroblasts, while cross-reactivity to epithelial cells was minimal. An aptamer-monomethyl auristatin F (MMAF) conjugate was generated by hybridizing with complementary DNA linked to MMAF. The resulting aptamer-MMAF conjugate inhibited proliferation of fibroblasts but appeared non-toxic to non-targeted epithelial cells. Our results show that artificial nucleic acid aptamer may potentially be used for fibroblast-specific therapy and diagnostic applications.

Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold.

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3'-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).

High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N3-Modified Thioflavin T Derivatives.

Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-( d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took ∼24 h for immunostaining to capture the corresponding fluorescence images.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

The investigation of protein-protein interactions (PPIs) and the preparation of antagonists are important for determining whether certain proteins are suitable medical targets. In the present study, we used the capillary electrophoresis-systematic evolution of ligands by exponential enrichment to generate natural and artificial nucleic acid aptamers targeting Ebola virus protein 24 (eVP24), demonstrating that artificial aptamers, synthesised utilising a uridine analogue with an adenine residue at its C5 position, exhibited activities exceeding those of natural ones. To confirm the functionality of the as-prepared aptamers, their abilities to inhibit the PPIs of eVP24 were determined by capillary electrophoresis and bio-layer interferometry, and the obtained results unambiguously demonstrated that these aptamers interacted with the functional site of eVP24 and were thus good antagonists.

Correction: Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

Correction for 'Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1' by Keisuke Tanaka et al., Org. Biomol. Chem., 2018, 16, 4456-4463.

Consecutive incorporation of functionalized nucleotides with amphiphilic side chains by novel KOD polymerase mutant.

Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.

Polymerase-mediated high-density incorporation of amphiphilic functionalities into DNA: enhancement of nuclease resistance and stability in human serum.

Modified oligodeoxyribonucleotides (mdODNs) bearing multiple copies of an amphiphilic functional group were enzymatically synthesized by simultaneous incorporation of base-modified 5'-triphosphate analogs of 2'-deoxyguanosine (dG(am)TP), 2'-deoxyuridine (dU(am)TP), 2'-deoxyadenosine (dA(am)TP), and 2'-deoxycytosine (dC(am)TP). The amphiphilic functionality, that is, (E)-38,53-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,52-diazapentapentacont-54-en-55-yl group, consists of the water soluble dodeca(ethylene glycol) chain and the hydrophobic dodecyl chain. An enzymatically synthesized ODN, composed of a 20-mer 5'-terminal segment containing 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotide (B/L nucleotide) and a 12-mer 3'-terminal segment containing the nucleobase-modified analogs, exhibits very high resistance against phosphodiesterase I and is stable in human serum for a longer period when compared with ODN, where the 12-mer 3'-terminal segment contains unmodified nucleotides.

Polymerase incorporation of a 2'-deoxynucleoside-5'-triphosphate bearing a 4-hydroxy-2-mercaptobenzimidazole nucleobase analogue.

Here, we describe the enzymatic construction of a new larger base pair formed between adenine (A) and a 4-hydroxy-2-mercaptobenzimidazole (SB) nucleobase analogue. We investigated the enzymatic incorporation of 2'-deoxynucleoside-5'-triphosphate bearing a SB nucleobase analogue (dSBTP) into oligonucleotides (ONs) by DNA polymerases. dSBTP could be effectively incorporated at the site opposite a dA in a DNA template by several B family DNA polymerases. These findings provide new insights into various aspects of biotechnology, including the design of non-natural base pairs.

Minimal thioflavin T modifications improve visual discrimination of guanine-quadruplex topologies and alter compound-induced topological structures.

We newly synthesized thioflavin T (ThT) analogs for which the methyl group at the N3 position on the benzothiazole ring was replaced with either a ((p-(dimethylamino)benzoyl)oxy)ethyl group (ThT-DB) or a hydroxyethyl group (ThT-HE). In several neutral buffers, ThT-HE bound to a parallel guanine-quadruplex (G4) DNA and selectively emitted strong fluorescence at 74- to 240-fold higher intensities than those in the presence of double-stranded DNA (dsDNA), whereas ThT resulted in only 13- to 25-fold higher intensities. Furthermore, circular dichroism (CD) analyses using ThT, ThT-DB, and ThT-HE showed that these compounds could induce topological changes in G4. In addition, the different chemical structures of the N3 substituents could alter a G4-DNA conformation. These results indicate a great potential for N3-substituted ThT analogs as G4 probes and drug leads to achieve gene expression regulation.