Copper-mediated siRNA activation for conditional control of gene expression.

Copper plays a crucial role in maintaining biological redox balance in living organisms, with elevated levels observed in cancer cells. Short interfering RNAs (siRNAs) are effective in gene silencing and find applications as both research tools and therapeutic agents. A method to regulate RNA interference using copper is especially advantageous for cancer-specific therapy. We present a chemical approach of selective siRNA activation triggered by intracellular copper ions. We designed and synthesized nucleotides containing copper-responsive moieties, which were incorporated into siRNAs. These copper-responsive siRNAs effectively silenced the target cyclin B1 mRNA in living cells. This pioneering study introduces a novel method for conditionally controlling gene silencing using biologically relevant metal ions in human cells, thereby expanding the repertoire of chemical knockdown tools.

RNA Oncological Therapeutics: Intracellular Hairpin RNA Assembly Enables MicroRNA-Triggered Anticancer Functionality.

RNA therapeutics are of global interest because of their versatility in targeting a variety of intracellular and extracellular biomolecules. In that context, long double-stranded RNA (dsRNA) has been studied as an antitumor agent that activates the immune response. However, its performance is constrained by poor cancer selectivity and cell-penetration ability. Here, we designed and synthesized an oncolytic RNA hairpin pair (oHP) that was selectively cytotoxic toward cancer cells expressing abundant oncogenic microRNA-21 (miR-21). Although the structure of each hairpin RNA was thermodynamically metastable, catalytic miR-21 input triggered it to open to generate a long nicked dsRNA. We demonstrated that oHP functioned as a cytotoxic amplifier of information in the presence of miR-21 in various cancer cells and tumor-bearing mice. This work represents the first example of the use of short RNA molecules as build-up-type anticancer agents that are triggered by an oncogenic miRNA.

Cellular Penetration and Intracellular Dynamics of Perfluorocarbon-Conjugated DNA/RNA as a Potential Means of Conditional Nucleic Acid Delivery.

Nucleic acid-based therapeutics represent a novel approach for controlling gene expression. However, a practical delivery system is required that overcomes the poor cellular permeability and intercellular instability of nucleic acids. Perfluorocarbons (PFCs) are highly stable structures that can readily traverse the lipid membrane of cells. Thus, PFC-DNA/RNA conjugates have properties that offer a potential means of delivering nucleic acid therapeutics, although the cellular dynamics of the conjugates remain unknown. Here, we performed systematic analysis of the cellular permeability of sequence-controlled PFC-DNA conjugates (N[PFC]n-DNA, n = 1,2,3,4,5) that can be synthesized by conventional phosphoramidite chemistry. We showed that DNA conjugates with two or more PFC-containing units (N[PFC]n≥2-DNA) penetrated HeLa cells without causing cellular damage. Imaging analysis along with quantitative flow cytometry analysis revealed that N[PFC]2-DNA rapidly passes through the cell membrane and is evenly distributed within the cytoplasm. Moreover, N[PFC]2-modified cyclin B1-targeting siRNA promoted gene knockdown efficacy of 30% compared with naked siRNA. A similar cell penetration without associated toxicity was consistent among the seven different human cell lines tested. These unique cellular environmental properties make N[PFC]2-DNA/RNA a potential nucleic acid delivery platform that can meet a wide range of applications.

Nucleic Acid-to-Small Molecule Converter through Amplified Hairpin DNA Circuits.

Many microRNAs (miRNAs) are characteristically found in cancer cells, making miRNAs promising marker biomolecules for cancer diagnosis and therapeutics. However, it is challenging to use miRNA as a cancer signature because it is difficult to convert the nucleic acid sequence information into molecular functionality. To address this challenge, we realize nucleic acid-to-small molecule converters using hairpin DNA circuits. Harnessing a Staudinger reduction as a trigger for the conversion, we constructed hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) circuits that respond to oncogenic miR-21. Fluorophore and dye molecules were released in response to miR-21 through the HCR, providing fluorogenic and chromogenic readouts. Selective cytotoxicity in miR-21-abundant cells was realized by the CHA to release the anticancer drug SN-38. This would be the first example of selective activation of a small-molecule prodrug triggered by oncogenic miRNA in human living cells.

Interstrand crosslinking oligonucleotides elucidate the effect of metal ions on the methylation status of repetitive DNA elements.

DNA methylation plays an important physiological function in cells, and environmental changes result in fluctuations in DNA methylation levels. Metal ions have become both environmental and health concerns, as they have the potential to disrupt the genomic DNA methylation status, even on specific sequences. In the current research, the methylation status of two typical repetitive DNA elements, i.e., long-interspersed nuclear element-1 (LINE-1) and alpha satellite (α-sat), was imaged and assessed using methylation-specific fluorescence in situ hybridization (MeFISH). This technique elucidated the effect of several metal ions on the methylation levels of repetitive DNA sequences. The upregulation and downregulation of the methylation levels of repetitive DNA elements by various metal ions were confirmed and depended on their concentration. This is the first example to investigate the effects of metal ions on DNA methylation in a sequence-specific manner.

Oncolytic Hairpin DNA Pair: Selective Cytotoxic Inducer through MicroRNA-Triggered DNA Self-Assembly.

Artificial nucleic acids have attracted much attention as potential cancer immunotherapeutic materials because they are recognized by a variety of extracellular and intracellular nucleic acid sensors and can stimulate innate immune responses. However, their low selectivity for cancer cells causes severe systemic immunotoxicity, making it difficult to use artificial nucleic acid molecules for immune cancer therapy. To address this challenge, we herein introduce a hairpin DNA assembly technology that enables cancer-selective immune activation to induce cytotoxicity. The designed artificial DNA hairpins assemble into long nicked double-stranded DNA triggered by intracellular microRNA-21 (miR-21), which is overexpressed in various types of cancer cells. We found that the products from the hairpin DNA assembly selectively kill miR-21-abundant cancer cells in vitro and in vivo based on innate immune activation. Our approach is the first to allow selective oncolysis derived from intracellular DNA self-assembly, providing a powerful therapeutic modality to treat cancer.

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.

anti-syn Unnatural Base Pair Enables Alphabet-Expanded DNA Self-Assembly.

Self-assembly properties and diversity in higher-order structures of DNA enable programmable tools to be used to construct algorithms at the molecular level. However, the utility of DNA-based programmable tools is hampered by the low orthogonality to natural nucleic acids, especially in complex molecular systems. To address this challenge, we report here the orthogonal regulation of DNA self-assembly by using an unnatural base pair (UBP) formation. Our newly designed UBP AnN:SyN is formed in combination with anti and unusual syn glycosidic conformation with high thermal stability and selectivity. Furthermore, AnC worked as a pH-sensitive artificial nucleobase, which forms a strong base pair with cytosine under a weak acidic condition (pH 6.0). The orthogonal AnN:SyN base pair functioned as a trigger for hybridization chain reaction to provide long nicked double-stranded DNA (ca. 1000 base pairs). This work represents the first example of the orthogonal DNA self-assembly that is nonreactive to natural four-letter alphabets DNA trigger and expands the types of programmable tools that work in a complex environment.

Floxuridine Oligomers Activated under Hypoxic Environment.

Floxuridine oligomers are anticancer oligonucleotide drugs composed of a number of floxuridine residues. They show enhanced cytotoxicity per floxuridine monomer because the nuclease degradation of floxuridine oligomers directly releases highly active floxuridine monophosphate in cells. However, their clinical use is limited by the low selectivity against cancer cells. To address this limitation, we herein report floxuridine oligomer prodrugs that are active under hypoxia conditions, which is one of the distinguishing features of the microenvironment of all solid tumors. We designed and synthesized two types of floxuridine oligomer prodrugs that possess hypoxia-responsive moieties on nucleobases. The floxuridine oligomer prodrugs showed lower cytotoxicity under normoxia conditions (O2 = 20%), while the parent floxuridine oligomer showed similar anticancer effects under hypoxia conditions (O2 = 1%). The floxuridine oligomer prodrug enabled tumor growth suppression in live mice. This would be the first example demonstrating the conditional control of the medicinal efficacy of oligomerized nucleoside anticancer drugs.

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.

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).

A highly constrained nucleic acid analog based on α-l-threosamine.

Chemically modified oligonucleotides (ONs) have recently gained much attention as therapeutic materials because of their improved properties. Here, a newly designed nucleic acid analog based on α-l-threosamine (named cTNA) is reported. cTNA has a "dual" constrained structure, with a bridged sugar moiety and shorter phosphoramidate backbone, to reduce the entropy loss during the hybridization. Unexpectedly, ONs containing the cTNA unit showed lower binding affinity with complementary RNA and DNA than natural ONs. Quantum chemical calculations imply that the relative nucleobase orientation of cTNA may be unfavorable for hybridization.

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation.

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.

Live-Cell Sensing of Telomerase Activity by Using Hybridization-Sensitive Fluorescent Oligonucleotide Probes.

Live-cell sensing of telomerase activity with simple and efficient strategies remains a challenging target. In this work, a strategy for telomerase sensing by using hybridization-sensitive fluorescent oligonucleotide probes is reported. In the presence of telomerase and dNTPs, the designed supporting strand was extended and generated the hairpin structure that catalyzed the next telomerase extending reaction. The special extension mechanism increased the local concentration of another supporting strand and telomerase, which resulted in enhanced telomerase activity. The hybridization-sensitive oligonucleotide probes bound to the hairpin catalyst and generated turn-on fluorescence. This method realized the sensing of telomerase activity in HeLa cell extract with a detection limit below 1.6×10-6  IU µL-1 . The real-time in situ observation of telomerase extension was achieved in living HeLa cells. This strategy has been applied to monitor the efficiency of telomerase-targeting anticancer drugs in situ.

A Hydrogen Peroxide Activatable Gemcitabine Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma.

The main concern in the use of anticancer chemotherapeutic drugs is host toxicity. Patients need to interrupt or change chemotherapy due to adverse effects. In this study, we aimed to decrease adverse events with gemcitabine (GEM) in the treatment of pancreatic ductal adenocarcinoma and focused on the difference of hydrogen peroxide levels in normal versus cancer cells. We designed and synthesized a novel boronate-ester-caged prodrug that is activated by the high H2 O2 concentrations found in cancer cells to release GEM. An H2 O2 -activatable GEM (A-GEM) has higher selectivity for H2 O2 over other reactive oxygen species (ROS) and cytotoxic effects corresponding to the H2 O2 concentration in vitro. A xenograft model of immunodeficient mice indicated that the effect of A-GEM was not inferior to that of GEM when administered in vivo. In particular, myelosuppression was significantly decreased following A-GEM treatment compared with that following GEM treatment.

Aryl Azides as Phosphine-Activated Switches for Small Molecule Function.

Engineered small molecule triggers are important tools for the control and investigation of biological processes, in particular protein function. Staudinger reductions of aryl azides to amines through the use of phosphines can trigger an elimination reaction, and thereby activation of a functional molecule, if an appropriately positioned leaving group is present. We conducted detailed investigations of the effect of aryl azide and phosphine structure on both the mechanism and kinetics of these reaction-induced eliminations and identified phosphine/azide pairs that enable complete activation within minutes under physiologically relevant conditions.

Hydrogen peroxide-triggered gene silencing in mammalian cells through boronated antisense oligonucleotides.

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) involved in various diseases, including neurodegeneration, diabetes, and cancer. Here, we introduce a new approach to use H2O2 to modulate specific gene expression in mammalian cells. H2O2-responsive nucleoside analogues, in which the Watson-Crick faces of the nucleobases are caged by arylboronate moieties, were synthesized. One of these analogues, boronated thymidine (dTB ), was incorporated into oligodeoxynucleotides (ODNs) using an automated DNA synthesizer. The hybridization ability of this boronated ODN to complementary RNA was clearly switched in the off-to-on direction upon H2O2 addition. Furthermore, we demonstrated H2O2-triggered gene silencing in mammalian cells using antisense oligonucleotides (ASOs) modified with dTB . Our approach can be used for the regulation of any gene of interest by the sequence design of boronated ASOs and will contribute to the development of targeted disease therapeutics.

Small Molecule Release and Activation through DNA Computing.

DNA-based logic gates can be assembled into computational devices that generate a specific output signal in response to oligonucleotide input patterns. The ability to interface with biological and chemical environments makes DNA computation a promising technology for monitoring cellular systems. However, DNA logic gate circuits typically provide a single-stranded oligonucleotide output, limiting the ability to effect biology. Here, we introduce a novel DNA logic gate design capable of yielding a small molecule output signal. Employing a Staudinger reduction as a trigger for the release and activation of a small molecule fluorophore, we constructed AND and OR logic gates that respond to synthetic microRNA (miRNA) inputs. Connecting the gates in series led to more complex DNA circuits that provided a small molecule output in response to a specific pattern of three different miRNAs. Moreover, our gate design can be readily multiplexed as demonstrated by simultaneous small molecule activation from two independent DNA circuits.

Synthesis and Properties of 7-Deazapurine- and 8-Aza-7-deazapurine-Locked Nucleic Acid Analogues: Effect of the Glycosidic Torsion Angle.

Conformationally restricted nucleoside analogues 2',4'-BNA/LNA-7-deazaguanine (LNA-7cG) and 2',4'-BNA/LNA-8-aza-7-deazaguanine (LNA-8n7cG), which avoid extra hydrogen bond formation at the 7-position of the guanine nucleobase, were successfully synthesized and incorporated into oligonucleotides. While the LNA-7cG-containing oligonucleotides show high duplex-forming ability with complementary DNA and RNA similar to LNA-G, the LNA-8n7cG-containing oligonucleotide has lower binding affinity than that of natural 2'-deoxyguanosine. This disparity in thermostability is also observed in 7-deazaadenosine analogues (LNA-7cA, LNA-8n7cA). Thermodynamic parameters and computational chemistry revealed that an inappropriate glycosidic torsion angle χ of 2',4'-BNA/LNA-8-aza-7-deazapurine analogues destabilizes duplex formation in contrast to 2',4'-BNA/LNA-7-deazapurine analogues. This result indicates that the nucleobase rotation angle plays an important role in duplex binding affinity. In addition, LNA-7cG-modified oligonucleotide effectively suppresses aggregation even in a guanine-rich sequence.

Biological applications of xeno nucleic acids.

Xeno nucleic acids (XNAs) are a group of chemically modified nucleic acid analogues that have been applied to various biological technologies such as antisense oligonucleotides, siRNAs and aptamers.