Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

Exosome-Hijacking Drug Delivery System with Branched Arginine Linker Effectively Deliver Antisense Oligonucleotides into Lung Adenocarcinoma Cells.

Exosomes are a type of extracellular vesicles that contain diverse molecules and are present in our body fluids. They play a crucial role in transporting materials and transmitting signals between cells. Currently, there have been numerous reports on the use of exosomes in drug delivery systems (DDS). However, most existing methods for utilizing exosomes in DDS require the isolation and purification of exosomes, which raises concerns about yield and potential damage to the exosomes. Recently, we have developed a novel DDS called "ExomiR-Tracker" that harnesses exosomes without the need for isolation and purification. This system aims to deliver nucleic acid drugs effectively. ExomiR-Tracker consists of an anti-exosome antibody equipped with nona-D-arginines (9 mer) and nucleic acid drugs which have complementary sequence of target microRNA (anti-miR). In this study, we modified ExomiR-Tracker by incorporating branched nona-D-arginines (9 + 9 mer) molecules (referred to as Branch ExomiR-Tracker) and evaluated its efficacy in lung adenocarcinoma cells (A549 cells). The improved complex formation ability and enhanced cellular uptake of anti-miR, demonstrated by our findings, highlight the advantages of incorporating branched oligoarginine peptides into the ExomiR-Tracker platform. These results represent significant progress in revealing the effectiveness of Branch ExomiR-Tracker against adhesive cancer cells, which has not been shown to be effective with the conventional Linear ExomiR-Tracker.

Synthesis and Evaluation of Oligonucleotide-Containing 2'-O-{[(4,5',8-Trimethylpsoralen)-4'-ylmethoxy]ethylaminocarb-onyl}adenosine as Photo-crosslinkable Gene Targeting Tools.

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-crosslinking properties of two Ps-Oligos, 5'-Ps-Oligo and a novel Ps-Oligo containing 2'-O-{[(4,5',8-trimethylpsoralen)-4'-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5'-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the interesting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies.

A Facile Method for the Quantification of Urinary Uracil Concentration by a Uracil-Specific Fluorescence Derivatization Reaction.

A facile and reliable fluorescence method for the quantification of urinary uracil concentration is proposed herein. The assay utilizes a specific fluorescence (FL) derivatization reaction for uracil using 3-methylbenzamidoxime as a fluorogenic reagent. Although the presence of urine inhibited the FL reaction, 10 µL of urine was sufficient for the detection of urinary uracil. The uracil derivative was successfully separated from other fluorescent impurities using simple reversed-phase LC with FL detection. Urinary uracil concentrations from 16 people were compared with the concentrations obtained by the traditional column-switching liquid chromatographic analysis with UV detection. The FL derivative of uracil appeared as a single peak in the chromatograms of all samples. However, several samples showed an additional peak overlapping the uracil peak when using the column-switching method because of UV-active impurities. These results indicated that that the present method is not affected by interfering substances in urine and affords a precise determination of urinary uracil. We expect the proposed method to be applicable for diagnosing dihydropyrimidine dehydrogenase deficiency in 5-fluorouracil chemotherapy.

Hydrazide Derivatives of Luminol for Chemiluminescence-Labelling of Macromolecules.

A series of chemiluminescent compounds containing a hydrazide group as a nucleophilic functional group has been synthesized. The syntheses were started from chemiluminescent luminol and isoluminol. The linker moiety was easily introduced onto non-nucleophilic exocyclic amino groups of luminol and isoluminol by gentle heating with cyclic acid anhydrides such as glutaric anhydride. The resulting carboxy group was converted to hydrazide by a simple condensation reaction using carbodiimide. Although majority of the synthesized compounds did not emit strong light, a sufficient chemiluminescence intensity was obtained from luminol-amido-C2-hydrazide (L2H) comprising of luminol scaffold with a dimethylene linker. The ability of L2H to form a covalent bond with a macromolecule was further investigated by incubation with oxidized horseradish peroxidase. The analysis on matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS revealed that the coupling efficiency of L2H was similar to that of commercially available labelling reagent having a hydrazide group. These results suggested that L2H, the luminol hydrazide containing a dimethylene linker, could be useful for the labelling of macromolecules in the sensitive bioassay such as chemiluminescence immunoassay.

Cross-Linking Antisense Oligodeoxyribonucleotides with a Photoresponsive α-Chloroaldehyde Moiety for RNA Point Mutations.

Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.

Development of Novel Antisense Oligonucleotides for the Functional Regulation of RNA-Induced Silencing Complex (RISC) by Promoting the Release of microRNA from RISC.

MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.

Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides.

We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation.

Rate-adjusted cross-linking reaction by photoresponsive α-bromoaldehyde (PBA)-conjugated ODN.

We developed a photoresponsive α-bromoaldehyde-conjugated oligonucleotide (PBA-ODN). The PBA-ODN selectively reacted and formed covalent bonds with target oligonucleotides having adenine or cytosine at the frontal position of the aldehyde derivative. Kinetic studies revealed that PBA-ODN has increased kinetic rates for the formation of cross-linked duplexes compared with the corresponding α-chloroaldehyde-conjugated oligonucleotide (PCA-ODN).

Dual-fluorescent RNA probes with an extremely large stokes shift.

Fluorescent probes are powerful and indispensable tools for imaging RNA in vivo and in vitro. To simultaneously visualize multiple RNA targets in a cell, it is necessary to develop probes which emit fluorescence with different colors by excitation at a single wavelength. We synthesized OMUpy1 and OMUpy2 in this study with a cyanine dye respectively conjugated at their 5' ends. A fluorescent analysis revealed these probes to have yellow or pink fluorescence derived from the cyanine dyes with an extremely large Stokes shift. Three color-coded fluorescent images were also obtained in the presence of target RNAs.

Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids.

Recent developments in artificial nucleic acid and drug delivery systems present possibilities for the symbiotic engineering of therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Employing these technologies, triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs) can be applied to the development of symbiotic genome-targeting tools as well as a new class of oligonucleotide drugs, which offer conceptual advantages over antisense as the antigene target generally comprises two gene copies per cell rather than multiple copies of mRNA that are being continually transcribed. Further, genome editing by TFOs or PNAs induces permanent changes in the pathological genes, thus facilitating the complete cure of diseases. Nuclease-based gene-editing tools, such as zinc fingers, CRISPR-Cas9, and TALENs, are being explored for therapeutic applications, although their potential off-target, cytotoxic, and/or immunogenic effects may hinder their in vivo applications. Therefore, this review is aimed at describing the ongoing progress in TFO and PNA technologies, which can be symbiotic genome-targeting tools that will cause a near-future paradigm shift in drug development.

Investigation of enhanced intracellular delivery of nanomaterials modified with novel cell-penetrating zwitterionic peptide-lipid derivatives.

Functionalized drug delivery systems have been investigated to improve the targetability and intracellular translocation of therapeutic drugs. We developed high functionality and quality lipids that met unique requirements, focusing on the quality of functional lipids for the preparation of targeted nanoparticles using microfluidic devices. While searching for a lipid with high solubility and dispersibility in solvents, which is one of the requirements, we noted that KK-(EK)4-lipid imparts nonspecific cellular association to polyethylene glycol (PEG)-modified (PEGylated) liposomes, such as cell-penetrating peptides (CPPs). We investigated whether KK-(EK)4-lipid, which has a near-neutral charge, is a novel CPP-modified lipid that enhances the intracellular translocation of nanoparticles. However, the cellular association mechanism of KK-(EK)4-lipid is unknown. Therefore, we synthesized (EK)n-lipid derivatives based on the sequence of KK-(EK)4-lipid and determined the sequence sites involved in cellular association. In addition, KK-(EK)4-lipid was applied to extracellular vesicles (EVs) and mRNA encapsulated lipid nanoparticles (mRNA-LNPs). KK-(EK)4-lipid-modified EVs and mRNA-LNPs showed higher cellular association and in vitro protein expression, respectively, compared to unmodified ones. We elucidated KK-(EK)4-lipid to have potential for applicability in the intracellular delivery of liposomes, EVs, and mRNA-LNPs.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged α-chloroaldehyde.

We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selective interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of α-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared for the synthesis of photoresponsive α-chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitrobenzyl)acetal group of a PCA-thymidine conjugate was completely removed by UV irradiation at 365 nm (400 mW/cm(2)) for 1 min. Photo-cross-linking studies revealed that PCA-ODN selectively reacted with the target nucleotides having an adenine or a cytosine moiety at the frontal position of the α-chloroaldehyde group.

RNA-based diagnosis in a multicellular specimen by whole mount in situ hybridization using an RNA-specific probe.

Recent RNA research has revealed the close involvement of various RNAs in cellular functions. RNAs are becoming the inevitable target molecules for research into details of gene expression. RNA and its related complexes are also promising targets for disease diagnosis. Multi cellular specimens such as organ tissues, histopathological specimens, and embryos are among the possible targets of RNA-based diagnostic techniques. In this report, we focused on a method that would provide such spatial and temporal information. We demonstrated that an RNA-specific probe (OMUpy2) was not only applicable to the detection of a specific mRNA in Drosophila embryos in a temporal and spatial manner but was also relatively quick and easy to use. The probe, OMUpy2, could be applied to other multi cellular systems for RNA-based diagnosis and research. The promising results of this manuscript show the great potential of RNA-based detection for both biological research and diagnostic medicine.

Therapeutic application of sequence-specific binding molecules for novel genome editing tools.

Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.

Chemistry of Therapeutic Oligonucleotides That Drives Interactions with Biomolecules.

Oligonucleotide therapeutics that can modulate gene expression have been gradually developed for clinical applications over several decades. However, rapid advances have been made in recent years. Artificial nucleic acid technology has overcome many challenges, such as (1) poor target affinity and selectivity, (2) low in vivo stability, and (3) classical side effects, such as immune responses; thus, its application in a wide range of disorders has been extensively examined. However, even highly optimized oligonucleotides exhibit side effects, which limits the general use of this class of agents. In this review, we discuss the physicochemical characteristics that aid interactions between drugs and molecules that belong to living organisms. By systematically organizing the related data, we hope to explore avenues for symbiotic engineering of oligonucleotide therapeutics that will result in more effective and safer drugs.

Successful Incorporation of Exosome-Capturing Antibody-siRNA Complexes into Multiple Myeloma Cells and Suppression of Targeted mRNA Transcripts.

Nucleic acid medicines have been developed as new therapeutic agents against various diseases; however, targeted delivery of these reagents into cancer cells, particularly hematologic cancer cells, via systemic administration is limited by the lack of efficient and cell-specific delivery systems. We previously demonstrated that monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers were transferred into solid cancer cells and inhibited exosomal miRNA functions. In this study, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNAs) with branched Arg linkers and investigated their effects on multiple myeloma (MM) cells. Anti-CD63 mAb-conjugated siRNAs were successfully incorporated into MM cells. The incorporation of exosomes was inhibited by endocytosis inhibitors. We also conducted a functional analysis of anti-CD63 mAb-conjugated siRNAs. Ab-conjugated luciferase+ (luc+) siRNAs significantly decreased the luminescence intensity in OPM-2-luc+ cells. Moreover, treatment with anti-CD63 mAb-conjugated with MYC and CTNNB1 siRNAs decreased the mRNA transcript levels of MYC and CTNNB1 to 52.5% and 55.3%, respectively, in OPM-2 cells. In conclusion, exosome-capturing Ab-conjugated siRNAs with branched Arg linkers can be effectively delivered into MM cells via uptake of exosomes by parental cells. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic cancers.

Microfluidic Post-Insertion Method for the Efficient Preparation of PEGylated Liposomes Using High Functionality and Quality Lipids.

Introduction: Targeted liposomes using ligand peptides have been applied to deliver therapeutic agents to the target sites. The post-insertion method is commonly used because targeted liposomes can be prepared by simple mixing of ligand peptide-lipid and liposomes. A large-scale preparation method is required for the clinical application of ligand-peptide-modified liposomes. Large-scale preparation involves an increase in volume and a change in the preparation conditions. Therefore, the physicochemical properties of liposomes may change owing to large alterations in the preparation conditions. To address this issue, we focused on a microfluidic device and developed a novel ligand peptide modification method, the microfluidic post-insertion method. Methods: We used integrin αvß3-targeted GRGDS (RGD) and cyclic RGDfK (cRGD)-modified high functionality and quality (HFQ) lipids, which we had previously developed. First, the preparation conditions of the total flow rate in the microfluidic device for modifying HFQ lipids to polyethylene glycol (PEG)-modified (PEGylated) liposomes were optimized by evaluating the physicochemical properties of the liposomes. The targeting ability of integrin αvß3-expressing colon 26 murine colorectal carcinoma cells was evaluated by comparing the cellular association properties of the liposomes prepared by the conventional post-insertion method. Results: When the RGD-HFQ lipid was modified into PEGylated liposomes by varying the total flow rate (1, 6, and 12 mL/min) of the microfluidic device, as the total flow rate increased, the polydispersity index also increased, whereas the particle size did not change. Furthermore, the RGD- and cRGD-modified PEGylated liposomes prepared at a total flow rate of 1 mL/min showed high cellular association properties equivalent to those prepared by the conventional post-insertion method. Conclusion: Microfluidic post-insertion method of HFQ lipids might be useful for clinical application and large-scale preparation of targeted liposomes.