Enzymatic synthesis of ligand-bearing oligonucleotides for the development of metal-responsive DNA materials.

Metal-mediated artificial base pairs are some of the most promising building blocks for constructing DNA-based supramolecules and functional materials. These base pairs are formed by coordination bonds between ligand-type nucleobases and a bridging metal ion and have been exploited to develop metal-responsive DNA materials and DNA-templated metal arrays. In this review, we provide an overview of methods for the enzymatic synthesis of DNA strands containing ligand-type artificial nucleotides that form metal-mediated base pairs. Conventionally, ligand-bearing DNA oligomers have been synthesized via solid-phase synthesis using a DNA synthesizer. In recent years, there has been growing interest in enzymatic methods as an alternative approach to synthesize ligand-bearing DNA oligomers, because enzymatic reactions proceed under mild conditions and do not require protecting groups. DNA polymerases are used to incorporate ligand-bearing unnatural nucleotides into DNA, and DNA ligases are used to connect artificial DNA oligomers to natural DNA fragments. Template-independent polymerases are also utilized to post-synthetically append ligand-bearing nucleotides to DNA oligomers. In addition, enzymatic replication of DNA duplexes containing metal-mediated base pairs has been intensively studied. Enzymatic methods facilitate the synthesis of DNA strands containing ligand-bearing nucleotides at both internal and terminal positions. Enzymatically synthesized ligand-bearing DNAs have been applied to metal-dependent self-assembly of DNA structures and the allosteric control of DNAzyme activity through metal-mediated base pairing. Therefore, the enzymatic synthesis of ligand-bearing oligonucleotides holds great potential in advancing the development of various metal-responsive DNA materials, such as molecular sensors and machines, providing a versatile tool for DNA supramolecular chemistry and nanotechnology.

Ligase-mediated synthesis of CuII-responsive allosteric DNAzyme with bifacial 5-carboxyuracil nucleobases.

A CuII-responsive allosteric DNAzyme has been developed by introducing bifacial 5-carboxyuracil (caU) nucleobases, which form both hydrogen-bonded caU-A and metal-mediated caU-CuII-caU base pairs. The base sequence was logically designed based on a known RNA-cleaving DNAzyme so that the caU-modified DNAzyme (caU-DNAzyme) can form a catalytically inactive structure containing three caU-A base pairs and an active form with three caU-CuII-caU pairs. The caU-DNAzyme was synthesized by joining short caU-containing fragments with a standard DNA ligase. The activity of caU-DNAzyme was suppressed without CuII, but enhanced 21-fold with the addition of CuII. Furthermore, the DNAzyme activity was turned on and off during the reaction by the addition and removal of CuII ions. Both ligase-mediated synthesis and CuII-dependent allosteric regulation were achieved by the bifacial base pairing properties of caU. This study provides a new strategy for designing stimuli-responsive DNA molecular systems.

DNA three-way junction structures with amino acid side chains prepared via post-synthetic amide condensation.

DNA three-way junction (3WJ) structures with three amino acid side chains in the core have been synthesized via post-synthetic DNA modification. Amide condensation reactions of oligonucleotides containing 2'-aminouridine with activated esters yielded DNA strands modified with His, Cys and Asp side chains to form modified 3WJs. Even a 3WJ with three negatively charged Asp side chains formed stably at room temperature. Furthermore, DNA hybridization alone placed two (His and Asp) and three (His, Cys, and Asp) side chains within the 3WJs, indicating that the DNA 3WJs are a useful platform for spatial arrangement of amino acid side chains.

Metal-dependent activity control of a compact-sized 8-17 DNAzyme based on metal-mediated unnatural base pairing.

A compact 8-17 DNAzyme was modified with a CuII-meditated artificial base pair to develop a metal-responsive allosteric DNAzyme. The base sequence was rationally designed based on the reported three-dimensional structure. The activity of the modified DNAzyme was enhanced 5.1-fold by the addition of one equivalent of CuII ions, showing good metal responsiveness. Since it has been challenging to modify compactly folded DNAzymes without losing their activity, this study demonstrates the utility of the metal-mediated artificial base pairing to create stimuli-responsive functional DNAs.

Metal-mediated DNA strand displacement and molecular device operations based on base-pair switching of 5-hydroxyuracil nucleobases.

Rational design of self-assembled DNA nanostructures has become one of the fastest-growing research areas in molecular science. Particular attention is focused on the development of dynamic DNA nanodevices whose configuration and function are regulated by specific chemical inputs. Herein, we demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner. The 5-hydroxyuracil (UOH) nucleobase is employed as a metal-responsive unit, forming both a hydrogen-bonded UOH-A base pair and a metal-mediated UOH-GdIII-UOH base pair. Metal-mediated strand displacement reactions are demonstrated under isothermal conditions based on the base-pair switching between UOH-A and UOH-GdIII-UOH. Furthermore, metal-responsive DNA tweezers and allosteric DNAzymes are developed as typical models for DNA nanodevices simply by incorporating UOH bases into the sequence. The metal-mediated base-pair switching will become a versatile strategy for constructing stimuli-responsive DNA nanostructures, expanding the scope of dynamic DNA nanotechnology.

Phenanthroline-modified DNA three-way junction structures stabilized by interstrand 3 : 1 metal complexation.

Incorporation of interstrand metal complexes into DNA is a versatile strategy for metal-dependent stabilization and structural induction of DNA supramolecular structures. In this study, we have synthesized DNA three-way junction (3WJ) structures modified with phenanthroline (phen) ligands. The phen-modified 3WJ was found to be thermally stabilized (ΔTm = +16.9 °C) by the formation of an interstrand NiII(phen)3 complex. Furthermore, NiII-mediated structure induction of 3WJs was demonstrated with the phen-modified strands and their unmodified counterparts. This study suggests that ligand-modified 3WJs would be useful structural motifs for the construction of metal-responsive DNA molecular systems.

Metal-dependent base pairing of bifacial iminodiacetic acid-modified uracil bases for switching DNA hybridization partner.

Dynamic control of DNA assembly by external stimuli has received increasing attention in recent years. Dynamic ligand exchange in metal complexes can be a central element in the structural and functional transformation of DNA assemblies. In this study, N,N-dicarboxymethyl-5-aminouracil (dcaU) nucleoside with an iminodiacetic acid (IDA) ligand at the 5-position of the uracil base has been developed as a bifacial nucleoside that can form both hydrogen-bonded and metal-mediated base pairs. Metal complexation study of dcaU nucleosides revealed their ability to form a 2:1 complex with a GdIII ion at the monomeric level. The characteristics of base pairing of dcaU nucleosides were then examined inside DNA duplexes. The results revealed that the formation of the metal-mediated dcaU-GdIII-dcaU pair significantly stabilized the DNA duplex containing one dcaU-dcaU mismatch (ΔT m = +16.1 °C). In contrast, a duplex containing a hydrogen-bonded dcaU-A pair was destabilized in the presence of GdIII (ΔT m = -3.5 °C). The GdIII-dependent base pairing of dcaU bases was applied to control the hybridization preference of DNA in response to metal ions. The hybridization partner of a dcaU-containing strand was reversibly exchanged by the addition and removal of GdIII ions. Since the incorporation of a single dcaU base can switch the hybridization behavior of DNA, the bifacial dcaU base would be a versatile building block for imparting metal responsiveness to DNA assemblies, allowing the rational design of dynamic DNA systems.

CuII-mediated DNA base pairing of a triazole-4-carboxylate nucleoside prepared by click chemistry.

Artificial metal-mediated DNA base pairing is a promising strategy for creating highly functionalized DNA supramolecules. Here we report a novel ligand-type triazole-4-carboxylate (TazC) nucleoside that is readily prepared by the click reaction. TazC nucleosides were found to form a stable TazC-CuII-TazC base pair inside DNA duplexes, resulting in CuII-specific duplex stabilization (ΔTm = +7.7 °C). This study demonstrates that the triazole derivatives are useful in the development of metal-mediated base pairing.

Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex.

Neonatal hypoxia causes cytotoxic neuronal swelling by the entry of ions and water. Multiple water pathways have been implicated in neurons because these cells lack water channels, and their membrane has a low water permeability. NKCC1 and KCC2 are cation-chloride cotransporters (CCCs) involved in water movement in various cell types. However, the role of CCCs in water movement in neonatal neurons during hypoxia is unknown. We studied the effects of modulating CCCs pharmacologically on neuronal swelling in the neocortex (layer IV/V) of neonatal mice (post-natal day 8-13) during prolonged and brief hypoxia. We used acute brain slices from Clomeleon mice which express a ratiometric fluorophore sensitive to Cl- and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl-]i by multiphoton microscopy. Neurons were identified using a convolutional neural network algorithm, and changes in the somatic area and [Cl-]i were evaluated using a linear mixed model for repeated measures. We found that (1) neuronal swelling and Cl- accumulation began after OGD, worsened during 20 min of OGD, or returned to baseline during reoxygenation if the exposure to OGD was brief (10 min). (2) Neuronal swelling did not occur when the extracellular Cl- concentration was low. (3) Enhancing KCC2 activity did not alter OGD-induced neuronal swelling but prevented Cl- accumulation; (4) blocking KCC2 led to an increase in Cl- accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation; (5) blocking NKCC1 reduced neuronal swelling during early but not prolonged OGD and aggravated Cl- accumulation during prolonged OGD; and (6) treatment with the "broad" CCC blocker furosemide reduced both swelling and Cl- accumulation during prolonged and brief OGD, whereas simultaneous NKCC1 and KCC2 inhibition using specific pharmacological blockers aggravated neuronal swelling during prolonged OGD. We conclude that CCCs, and other non-CCCs, contribute to water movement in neocortical neurons during OGD in the neonatal period.

CuII-mediated stabilisation of DNA duplexes bearing consecutive ethenoadenine lesions and its application to a metal-responsive DNAzyme.

Metal-mediated nucleobase pairing can play a central role in the expression of metal-responsive DNA functions. We report the CuII-mediated stabilisation of DNA duplexes bearing damaged nucleobases, 1,N6-ethenoadenine (εA), as metal-binding sites, which was utilised to construct a metal-responsive DNAzyme. Consecutive incorporation of three or more εA-εA mismatch pairs allowed for CuII-dependent significant duplex stabilisation through metal-mediated εA-CuII-εA base pairing. Subsequently, a split DNAzyme with three εA-CuII-εA base pairs was strategically designed. The activity of the εA-modified DNAzyme was enhanced by 5.3-fold upon addition of CuII ions. This study demonstrates the utility of εA lesions for building metal-responsive DNA architectures.

Multiple Cerebral Hemorrhages and White Matter Lesions Developing after Severe hMPV Pneumonia in a Patient with Trisomy 13: A Case Report and Review of the Literature.

Human metapneumovirus (hMPV) is a common cause of upper and lower respiratory tract infections in children. A few case reports have described hMPV encephalitis or encephalopathy. Neuroimaging data on patients with hMPV encephalitis are scarce. We report a patient with trisomy 13 who developed severe hMPV pneumonia, multifocal cerebral and cerebellar hemorrhagic infarctions and extensive cerebral white matter demyelination. Although adult respiratory distress syndrome and disseminated intravascular coagulation contributed to the devastating central nervous system (CNS) lesions, endothelial dysfunction of the CNS caused by hMPV infection probably also played a pathophysiological role in this case.

Metal-mediated DNA base pairing of easily prepared 2-oxo-imidazole-4-carboxylate nucleotides.

Metal-mediated DNA base pairs, which consist of two ligand-type artificial nucleobases and a bridging metal ion, have attracted increasing attention in recent years as a different base pairing mode from natural base pairing. Metal-mediated base pairing has been extensively studied, not only for metal-dependent thermal stabilisation of duplexes, but also for metal assembly by DNA templates and construction of functional DNAs that can be controlled by metals. Here, we report the metal-mediated base paring properties of a novel 2-oxo-imidazole-4-carboxylate (ImOC) nucleobase and a previously reported 2-oxo-imidazole-4-carboxamide (ImOA) nucleobase, both of which can be easily derived from a commercially available uridine analogue. The ImOC nucleobases were found to form stable ImOC-CuII-ImOC and ImOC-HgII-ImOC base pairs in the presence of the corresponding metal ions, leading to an increase in the duplex melting temperature by +20 °C and +11 °C, respectively. The ImOC bases did not react with other divalent metal ions and showed superior metal selectivity compared to similar nucleobase design reported so far. The ImOC-CuII-ImOC base pair was much more stable than mismatch pairs with other natural nucleobases, confirming the base pair specificity in the presence of CuII. Furthermore, we demonstrated the quantitative assembly of three CuII ions inside a DNA duplex with three consecutive ImOC-ImOC pairs, showing great potential of DNA-template based CuII nanoarray construction. The study of easily-prepared ImOC base pairs will provide a new design strategy for metal-responsive DNA materials.

Primary pyomyositis in an infant following one-day afebrile upper limb monoplegia.

Background : Pyomyositis is a subacute bacterial infection of the skeletal muscles. Its most common features are fever, muscle swelling, and focal pain. There have been insufficient data regarding pyomyositis in healthy infants. Case report : A one-month-old boy presented with an impairment of his left arm movement. He was well-nourished and not under any apparent distress. His vital signs were within the normal limits but neurological examination revealed left forearm paralysis. Physical examination showed no abnormal findings in the region from the left shoulder joint to the fingertips. Considering these factors, an intracranial pathology was initially suspected. However, he developed a fever, regular tachycardia, and swelling in the left forearm. Magnetic resonance imaging revealed inflammation in the left forearm muscles. He was diagnosed with bacterial myositis and started on intravenous antibiotics. On the 17th day, he was discharged with oral antibiotic treatment, which was completed over 25 days without any sequelae nor relapse. Conclusion : Here we report the case of Japanese primary pyomyositis following one-day afebrile upper limb monoplegia in an infant. Even when infants exhibit afebrile symptoms, a bacterial infection should be suspected. J. Med. Invest. 68 : 372-375, August, 2021.

Bipyridine-Modified DNA Three-Way Junctions with Amide Linkers: Metal-Dependent Structure Induction and Self-Sorting.

Invited for the cover of this issue are Yusuke Takezawa, Shiori Sakakibara, and Mitsuhiko Shionoya at The University of Tokyo. The image depicts the formation of stable DNA three-way junction structures crosslinked by an interstrand NiII (bpy)3 complex. Read the full text of the article at 10.1002/chem.202104037.

Bipyridine-Modified DNA Three-Way Junctions with Amide linkers: Metal-Dependent Structure Induction and Self-Sorting.

DNA three-way junction (3WJ) structures are essential building blocks for the construction of DNA nanoarchitectures. We have synthesized a bipyridine (bpy)-modified DNA 3WJ by using a newly designed bpy-modified nucleoside, Ubpy -3, in which a bpy ligand is tethered via a stable amide linker. The thermal stability of the bpy-modified 3WJ was greatly enhanced by the formation of an interstrand NiII (bpy)3 complex at the junction core (ΔTm =+17.7 °C). Although the stereochemistry of the modification site differs from that of the previously reported bpy-modified nucleoside Ubpy -2, the degree of the NiII -mediated stabilization observed with Ubpy -3 was comparable to that of Ubpy -2. Structure induction of the 3WJs and the duplexes was carried out by the addition or removal of NiII ions. Furthermore, NiII -mediated self-sorting of 3WJs was performed by using the bpy-modified strands and their unmodified counterparts. Both transformations were driven by the formation of NiII (bpy)3 complexes. The structural induction and self-sorting of bpy-modified 3WJs are expected to have many potential applications in the development of metal-responsive DNA materials.

Metal-responsive reversible binding of triplex-forming oligonucleotides with 5-hydroxyuracil nucleobases.

Metal-responsive triplex-forming oligonucleotides (TFOs) were synthesised by incorporating 5-hydroxyuracil (UOH) nucleobases as metal recognition sites. Binding of the UOH-containing TFO to the target natural DNA duplexes was reversibly regulated by the addition and removal of GdIII ions under isothermal conditions.

Site-specific polymerase incorporation of consecutive ligand-containing nucleotides for multiple metal-mediated base pairing.

An enzymatic method has been developed for the synthesis of DNA oligomers containing consecutive artificial ligand-type nucleotides. Three hydroxypyridone ligand-containing nucleotides forming CuII-mediated unnatural base pairs were continuously incorporated at a pre-specified position by a lesion-bypass Dpo4 polymerase. This enzymatic synthesis was applied to the development of a CuII-responsive DNAzyme. Accordingly, this research will open new routes for the construction of metal-responsive DNA architectures that are manipulated by multiple metal-mediated base pairing.

Metal-Dependent DNA Base Pairing of 5-Carboxyuracil with Itself and All Four Canonical Nucleobases.

A 5-carboxyuracil (caU) nucleobase was found to pair not only with A (caU-A) by hydrogen bonding but also with other DNA nucleobases by metal coordination bonding. Metal-dependent formation of caU-CuII-caU, caU-HgII-T, caU-AgI-C, and caU-CuII-G pairs was demonstrated by duplex melting analysis and mass spectrometry. The duplexes containing caU-X (X = caU, T, C, and G) were significantly stabilized in the presence of the corresponding metal ions, while the DNA duplexes containing the caU-A pairs were destabilized by the addition of CuII. These results suggest that the hybridization partner of caU-containing DNA strands can be altered by metal complexation. As a result, this study provides a new direction to integrate caU nucleobases to construct diverse metallo-DNA supramolecules and metal-responsive DNA devices.

Sharp Switching of DNAzyme Activity through the Formation of a CuII -Mediated Carboxyimidazole Base Pair.

DNAzymes are widely used as functional units for creating DNA-based sensors and devices. Switching of DNAzyme activity by external stimuli is of increasing interest. Herein we report a CuII -responsive DNAzyme rationally designed by incorporating one of the most stabilizing artificial metallo-base pairs, a CuII -mediated carboxyimidazole base pair (ImC -CuII -ImC ), into a known RNA-cleaving DNAzyme. Cleavage of the substrate was suppressed without CuII , but the reaction proceeded efficiently in the presence of CuII ions. This is due to the induction of a catalytically active structure by ImC -CuII -ImC pairing. The on/off ratio was as high as 12-fold, which far exceeds that of the previously reported DNAzyme with a CuII -mediated hydroxypyridone base pair. The DNAzyme activity can be regulated specifically in response to CuII ions during the reaction through the addition, removal, or reduction of CuII . This approach should advance the development of stimuli-responsive DNA systems with a well-defined sharp switching function.

Correction: Biallelic GALM pathogenic variants cause a novel type of galactosemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.