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.

An osteoinductive surface by adhesive bone morphogenetic protein-2 prepared using the bioorthogonal approach for tight binding of titanium with bone.

Inorganic biomaterials are used in various orthopedic and dental implants. Nevertheless, they cause clinical issues such as loosening of implants and patient morbidity. Therefore, inspired by mussel adhesive proteins, we aimed to design an adhesive and dimer-forming highly active bone morphogenetic protein-2 (BMP-2) using bioorthogonal chemistry, in which recombinant DNA technology was combined with enzymatic modifications, to achieve long-term osseointegration with titanium. The prepared BMP-2 exhibited substantially higher binding activity than wild-type BMP-2, while the adhered BMP-2 was more active than soluble BMP-2. Therefore, the adhesive BMP-2 was immobilized onto titanium wires and screws and implanted into rat bones, and long-term osteogenesis was evaluated. Adhesive BMP-2 promoted the mechanical binding of titanium to bones, enabling efficient bone regeneration and effective stabilization of implants. Thus, such adhesive biosignaling proteins can be used in regenerative medicine.

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.

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization.

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain.

Heterotypic polyubiquitins are an emerging class of polyubiquitins that have attracted interest because of their potential diversity of structures and physiological functions. There is an increasing demand for structure-defined synthesis of heterotypic chains to investigate the topological factors underlying the intracellular signals that are characteristically mediated by the heterotypic chain. However, the applicability of chemical and enzymatic polyubiquitin synthesis developed to date has been limited by laborious rounds of ligation and purification or by a lack of modularity of the chain structure with respect to the length and the branch position. Here, we established a one-pot, photocontrolled synthesis of structurally defined heterotypic polyubiquitin chains. We designed ubiquitin derivatives with a photolabile protecting group at a lysine residue used for polymerization. Repetitive cycles of linkage-specific enzymatic elongation and photoinduced deprotection of the protected ubiquitin units enabled stepwise addition of ubiquitins with appropriate functionalities to control the length and branching positions. The positional control of branching was achieved without isolation of intermediates, allowing one-pot synthesis of K63 triubiqutin chains and a K63/K48 heterotypic tetraubiquitin chain with defined branching positions. The present study provides a chemical platform for the efficient construction of long polyubiquitin chains with defined branch structures that will facilitate the understanding of the essential relationships between functions and structures of the heterotypic chain that have hitherto been overlooked.

Cytosolic protein delivery using ultrasound-guided vaporization of perfluorocarbon nano-droplets.

Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins.

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.

cIAP1-based degraders induce degradation via branched ubiquitin architectures.

Targeted protein degradation through chemical hijacking of E3 ubiquitin ligases is an emerging concept in precision medicine. The ubiquitin code is a critical determinant of the fate of substrates. Although two E3s, CRL2VHL and CRL4CRBN, frequently assemble with proteolysis-targeting chimeras (PROTACs) to attach lysine-48 (K48)-linked ubiquitin chains, the diversity of the ubiquitin code used for chemically induced degradation is largely unknown. Here we show that the efficacy of cIAP1-targeting degraders depends on the K63-specific E2 enzyme UBE2N. UBE2N promotes degradation of cIAP1 induced by cIAP1 ligands and subsequent cancer cell apoptosis. Mechanistically, UBE2N-catalyzed K63-linked ubiquitin chains facilitate assembly of highly complex K48/K63 and K11/K48 branched ubiquitin chains, thereby recruiting p97/VCP, UCH37 and the proteasome. Degradation of neo-substrates directed by cIAP1-recruiting PROTACs also depends on UBE2N. These results reveal an unexpected role for K63-linked ubiquitin chains and UBE2N in degrader-induced proteasomal degradation and demonstrate the diversity of the ubiquitin code used for chemical hijacking.

Bioorthogonal Photoreactive Surfaces for Single-Cell Analysis of Intercellular Communications.

Methods to construct single-cell pairs of heterogeneous cells attract attention because of their potential in cell biological and medical applications for analyzing individual intercellular communications such as immune and nerve synaptic interactions. Photoactivatable substrate surfaces for cell anchoring are promising tools to achieve single-cell pairing. However, conventional surfaces that photoactivate a single type of cell anchoring moiety restrict the combination of cell pair types and their applications. We developed a photoresponsive material comprising a bioorthogonal photoreactive moiety and non-cell adhesive hydrophilic polymer. The material-coated surface allows conjugation with various cell anchoring molecules in response to light at specific timings and consequently achieves light-induced anchoring of a variety of cells at defined regions. Using the platform surface, an array of cancer cell and natural-killer (NK) cell pairs was constructed on a flat substrate surface and the dynamic morphological changes of the cancer cells were monitored by cytotoxic interaction with NK cells at a single-cell level. The photoreactive surface is a useful tool for image-based investigation of the communications between a variety of cell types.

Intracellular Protein Photoactivation Using Sterically Bulky Caging.

Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both in vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.

Chemoenzymatic Labeling to Visualize Intercellular Contacts Using Lipidated SortaseA.

Methods to label intercellular contact have attracted attention because of their potential in cell biological and medical applications for the analysis of intercellular communications. In this study, a simple and versatile method for chemoenzymatic labeling of intercellularly contacting cells is demonstrated using a cell-surface anchoring reagent of a poly(ethylene glycol)(PEG)-lipid conjugate. The surface of each cell in the cell pairs of interest were decorated with sortase A (SrtA) and triglycine peptide that were lipidated with PEG-lipid. In the mixture of the two-cell populations, the triglycine-modified cells were enzymatically labeled with a fluorescent labeling reagent when in contact with SrtA-modified cells on a substrate. The selective labeling of the contacting cells was confirmed by confocal microscopy. The method is a promising tool for selective visualization of intercellularly contacting cells in cell mixtures for cell-cell communication analysis.

Repetitive Thiazolidine Deprotection Using a Thioester-Compatible Aldehyde Scavenger for One-Pot Multiple Peptide Ligation.

Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N',N'-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors.

Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

Photoactivatable Materials for Versatile Single-Cell Patterning Based on the Photocaging of Cell-Anchoring Moieties through Lipid Self-Assembly.

Versatile methods for patterning multiple types of cells with single-cell resolution have become an increasingly important technology for cell analysis, cell-based device construction, and tissue engineering. Here, we present a photoactivatable material based on poly(ethylene glycol) (PEG)-lipids for patterning a variety of cells, regardless of their adhesion abilities. In this study, PEG-lipids bearing dual fatty acid chains were first shown to perfectly suppress cell anchoring on their coated substrate surfaces whereas those with single-chain lipids stably anchored cells through lipid-cell membrane interactions. From this finding, a PEG-lipid with one each of both normal and photocleavable fatty acid chains was synthesized as a material that could convert the chain number from two to one by exposure to light. On the photoconvertible PEG-lipid surface, cell anchoring was activated by light exposure. High-speed atomic force microscopy measurements revealed that this photocaging of the lipid-cell membrane interaction occurs because the hydrophobic dual chains self-assemble into nanoscale structures and cooperatively inhibit the anchoring. Light-induced dissociation of the lipid assembly achieved the light-guided fine patterning of multiple cells through local photoactivation of the anchoring interactions. Using this surface, human natural killer cells and leukemia cells could be positioned to interact one-by-one. The cytotoxic capacity of single immune cells was then monitored via microscopy, showing the proof-of-principle for applications in the high-throughput analysis of the heterogeneity in individual cell-cell communications. Thus, the substrate coated with our photoactivatable material can serve as a versatile platform for the accurate and rapid patterning of multiple-element cells for intercellular communication-based diagnostics.

Changes of C≡C Triple Bond Vibration that Disclosed Non-Canonical Cytosine Protonation in i-Motif-Forming Oligodeoxynucleotides.

Non-canonical protonation at cytosine (C) in DNA is related to a formation of second order DNA structures such as i-motif, which has a role in gene regulation. Although the detailed structural information is indispensable for comprehension of their functions in cells, the protonation status of C in complicated environments is still elusive. To provide a reporter system of non-canonical protonation, we focused on the molecular vibration that could be monitored using the Raman spectroscopy. We prepared a cytosine derivative (PC) with an acetylene unit as a Raman tag, and found that the Raman signal of acetylene in PC in oligodeoxynucleotides (ODNs) changed due to protonation at the cytosine ring which shortened an acetylene bond. The signal change in i-motif-forming ODNs was also observed in crowded environments with polyethylene glycol, evidencing protonation in i-motif DNA in complicated environments. This system would be one of tracking tools for protonation in DNA structures.

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.

Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest.

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.