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.

Polyamines promote xenobiotic nucleic acid synthesis by modified thermophilic polymerase mutants.

The enzymatic synthesis of xenobiotic nucleic acids (XNA), which are artificially sugar-modified nucleic acids, is essential for the preparation of XNA libraries. XNA libraries are used in the in vitro selection of XNA aptamers and enzymes (XNAzymes). Efficient enzymatic synthesis of various XNAs can enable the screening of high-quality XNA aptamers and XNAzymes by expanding the diversity of XNA libraries and adding a variety of properties to XNA aptamers and XNAzymes. However, XNAs that form unstable duplexes with DNA, such as arabino nucleic acid (ANA), may dissociate during enzyme synthesis at temperatures suitable for thermophilic polymerases. Thus, such XNAs are not efficiently synthesised by the thermophilic polymerase mutants at the end of the sequence. This undesirable bias reduces the possibility of generating high-quality XNA aptamers and XNAzymes. Here, we demonstrate that polyamine-induced DNA/ANA duplex stabilisation promotes ANA synthesis that is catalysed by thermophilic polymerase mutants. Several polyamines, including spermine, spermidine, cadaverine, and putrescine promote ANA synthesis. The negative effect of polyamines on the fidelity of ANA synthesis was negligible. We also showed that polyamines promote the synthesis of other XNAs, including 2'-amino-RNA/2'-fluoro-RNA mixture and 2'-O-methyl-RNA. In addition, we found that polyamine promotes DNA synthesis from the 2'-O-methyl-RNA template. Polyamines, with the use of thermophilic polymerase mutants, may allow further development of XNA aptamers and XNAzymes by promoting the transcription and reverse transcription of XNAs.

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.

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.

Light-controllable cell-membrane disturbance for intracellular delivery.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

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.

Inhibition of microRNA-33b in humanized mice ameliorates nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.

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.

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.

Inhibition of microRNA-33b specifically ameliorates abdominal aortic aneurysm formation via suppression of inflammatory pathways.

Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA.

Administration of Gapmer-type Antisense Oligonucleotides Targeting γ-Glutamylcyclotransferase Suppresses the Growth of A549 Lung Cancer Xenografts.

BACKGROUND/AIM: γ-Glutamyl cyclotransferase (GGCT) is up-regulated in various cancer types, including lung cancer. In this study, we evaluated efficacy of gapmer-type antisense oligonucleotides (ASOs) targeting GGCT in an A549 lung cancer xenograft mouse model and studied their mechanisms of action. MATERIALS AND METHODS: GGCT was inhibited using GGCT-ASOs and cell proliferation was evaluated by dye exclusion test. Western blot analysis was conducted to measure expression of GGCT, p21, p16 and p27, phosphorylation of AMP-activated protein kinase, and caspase activation in A549 cells. Induction of apoptosis and up-regulation of reactive oxygen species were assessed by flow cytometry using annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate dye, respectively. RESULTS: GGCT-ASOs suppressed GGCT expression in A549 cells, inhibited proliferation, and induced apoptosis with activation of caspases. GGCT-ASOs also increased expression of cell-cycle regulating proteins, phospho-AMPK and ROS levels. Systemic administration of GGCT-ASOs to animals bearing A549 lung cancer xenografts showed significant antitumor effects without evident toxicity. CONCLUSION: GGCT-ASOs appear to be promising as novel cancer therapeutic agents.

Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity.

Nucleic acid aptamers have attracted considerable attention as next-generation pharmaceutical agents and delivery vehicles for small molecule drugs and therapeutic oligonucleotides. Chemical modification is an effective approach for improving the functionality of aptamers. However, the process of selecting appropriately modified aptamers is laborious because of many possible modification patterns. Here, we describe a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) approach for the generation of the artificial nucleic acid aptamers effective against human TROP2, a cell surface protein identified by drug discovery as a promising target for cancer therapy. Capillary electrophoresis SELEX was used for the pre-screening of multiple modified nucleic acid libraries and enrichment of TROP2 binding aptamers in the first step, followed by functional screening using cell-SELEX in the second step for the generation of cell-internalizing aptamers. One representative aptamer, Tac-B1, had a nanomolar-level affinity to human TROP2 and exhibited elevated capacity for internalization by cells. Because of the growing interest in the application of aptamers for drug delivery, our hybrid selection approach has great potential for the generation of functional artificial nucleic acid aptamers with ideal modification patterns in vitro.

Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide.

Intracellular delivery of oligonucleotides is important for their use as therapeutic drugs. The conjugation of molecules interacting with cell membrane proteins to enhance their internalization into cells is an effective strategy for delivering oligonucleotides. In the present study, we focused on creating aptamers, which are single-stranded oligonucleotides that bind target molecules with high affinity and specificity, as membrane protein-binding molecules. With an evolutionary selection approach using a random DNA library containing a uracil derivative with a hydrophobic functional group at the 5 position, we successfully obtained aptamers that are efficiently internalized into A549 cells. The efficacies of the aptamers were tested by further conjugation with MALAT1-targeting antisense oligonucleotides (ASOs), and the expression levels of MALAT1 RNA were examined. The aptamer-ASO conjugates were taken up by A549 cells, although there was no observable reduction in MALAT1 RNA levels. In contrast, the activity of the aptamer-ASO conjugate was potentiated when endosomal/lysosomal escape was enhanced by the addition of chloroquine. Thus, we showed that the hydrophobic modification of the nucleobase moiety is useful for developing highly internalizing aptamers and that endosomal/lysosomal escape is important for the intracellular delivery of ASOs by aptamers.

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.

Amido-Bridged Nucleic Acid-Modified Antisense Oligonucleotides Targeting SYT13 to Treat Peritoneal Metastasis of Gastric Cancer.

Patients with peritoneal metastasis of gastric cancer have dismal prognosis, mainly because of inefficient systemic delivery of drugs to peritoneal tumors. We aimed to develop an intraperitoneal treatment strategy using amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides (ASOs) targeting synaptotagmin XIII (SYT13) and to identify the function of SYT13 in gastric cancer cells. We screened 71 candidate oligonucleotide sequences according to SYT13-knockdown efficacy, in vitro activity, and off-target effects. We evaluated the effects of SYT13 knockdown on cellular functions and signaling pathways, as well as the effects of intraperitoneal administration to mice of AmNA-modified anti-SYT13 ASOs. We selected the ASOs (designated hSYT13-4378 and hSYT13-4733) with the highest knockdown efficiencies and lowest off-target effects and determined their abilities to inhibit cellular functions associated with the metastatic potential of gastric cancer cells. We found that SYT13 interfered with focal adhesion kinase (FAK)-mediated intracellular signals. Intraperitoneal administration of hSYT13-4378 and hSYT13-4733 in a mouse xenograft model of metastasis inhibited the formation of peritoneal nodules and significantly increased survival. Reversible, dose- and sequence-dependent liver damage was induced by ASO treatment without causing abnormal morphological and histological changes in the brain. Intra-abdominal administration of AmNA-modified anti-SYT13 ASOs represents a promising strategy for treating peritoneal metastasis of gastric cancer.

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).

Inhibiting guanine oxidation and enhancing the excess-electron-transfer efficiency of a pyrene-modified oligonucleotide by introducing an electron-donating group on pyrene.

The introduction of an electron-donating group (e.g., methoxy or piperidinyl) onto the pyrene moiety of 5-(pyrenylethynyl)-2'-deoxyuridine (PyU) increases its DNA-mediated reductive electron-transfer efficiency. In addition, these modifications dramatically inhibit photoinduced guanine oxidation.

GREB1 induced by Wnt signaling promotes development of hepatoblastoma by suppressing TGFß signaling.

The ß-catenin mutation is frequently observed in hepatoblastoma (HB), but the underlying mechanism by which Wnt/ß-catenin signaling induces HB tumor formation is unknown. Here we show that expression of growth regulation by estrogen in breast cancer 1 (GREB1) depends on Wnt/ß-catenin signaling in HB patients. GREB1 is localized to the nucleus where it binds Smad2/3 in a competitive manner with p300 and inhibits TGFß signaling, thereby promoting HepG2 HB cell proliferation. Forced expression of ß-catenin, YAP, and c-Met induces HB-like mouse liver tumor (BYM mice), with an increase in GREB1 expression and HB markers. Depletion of GREB1 strongly suppresses marker gene expression and HB-like liver tumorigenesis, and instead enhances TGFß signaling in BYM mice. Furthermore, antisense oligonucleotides for GREB1 suppress the formation of HepG2 cell-induced tumors and HB-like tumors in vivo. We propose that GREB1 is a target molecule of Wnt/ß-catenin signaling and required for HB progression.