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.

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.

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.

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.

Allosteric Regulation of DNAzyme Activities through Intrastrand Transformation Induced by Cu(II)-Mediated Artificial Base Pairing.

Allosteric regulation is gaining increasing attention as a basis for the production of stimuli-responsive materials in many research areas including DNA nanotechnology. We expected that metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, could serve as allosteric units that regulate the function of DNA molecules. In this study, we established a rational design strategy for developing CuII-responsive allosteric DNAzymes by incorporating artificial hydroxypyridone ligand-type nucleotides (H) that form a CuII-mediated base pair (H-CuII-H). We devised a new enzymatic method using a standard DNA polymerase and a ligase to prepare DNA strands containing H nucleotides. Previously reported DNAzymes were modified by introducing a H-H pair into the stem region, and the stem-loop sequences were altered so that the structure becomes catalytically inactive in the absence of CuII ions. The formation of a H-CuII-H base pair triggers intrastrand transformation from the inactive to the active structure, enabling allosteric regulation of the DNAzyme activity in response to CuII ions. The activity of the H-modified DNAzyme was reversibly switched by the addition and removal of CuII ions under isothermal conditions. Similarly, by incorporating a H-CuII-H pair into an in vitro-selected AgI-dependent DNAzyme, we have developed a DNAzyme that exhibits an AND logic-gate response to CuII and AgI ions. The rational design strategy and the easy enzymatic synthetic method presented here provide a versatile way to develop a variety of metal-responsive allosteric DNA materials, including molecular machines and logic circuits, based on metal-mediated artificial 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.

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.

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.

Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides.

Metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, have shown promise as functional units to develop stimuli-responsive DNA materials. Although a variety of metal-mediated base pairs have been constructed with artificial ligand-type nucleotides and various metal ions, the application of such metal-mediated base pairs has been relatively poorly explored mainly due to the cumbersome chemical synthesis of artificial DNA strands. Herein we report a facile enzymatic method to synthesize DNA strands containing a ligand-type hydroxypyridone (H) nucleotide, which forms a CuII-mediated base pair (H-CuII-H). A two-step primer extension reaction using two commercially available polymerases enabled the incorporation of a H nucleotide at an internal position of oligonucleotides. The polymerase synthesis was subsequently applied to the development of metal-responsive deoxyribozymes (DNAzymes), whose catalytic activity was regulated by the formation of a single H-CuII-H base pair in its stem region. The DNAzyme activity was reversibly switched by the alternate addition and the removal of CuII ions. Furthermore, metal-dependent orthogonal activation of a CuII-responsive H-DNAzyme and a HgII-responsive T-DNAzyme was experimentally demonstrated by utilizing both H-CuII-H as well as widely explored T-HgII-T base pairs. These results suggest that the incorporation of H-CuII-H base pairs would facilitate the rational design of metal-responsive functional DNAs. Accordingly, the facile enzymatic synthesis of artificial ligand-bearing DNAs developed in this study would significantly expand the toolbox of DNA-based supramolecular chemistry and DNA nanotechnology.

Biallelic GALM pathogenic variants cause a novel type of galactosemia.

PURPOSE: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. METHODS: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. RESULTS: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between ß- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. CONCLUSION: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.

Leucine-485 deletion variant of BRAF may exhibit the severe end of the clinical spectrum of CFC syndrome.

The genotype-phenotype correlation in BRAF variant in cardio-facio-cutaneous (CFC) syndrome is not clearly defined. Here we report a case with a severe clinical phenotype and a novel BRAF variant, p.Leu485del. The present case showed severe intellectual disability, impaired awareness, hyperekplexia, involuntary movements, early onset refractory seizures, and delayed myelination on brain magnetic resonance imaging as well as a polycystic and dysplastic kidney, which are previously unreported anomalies in CFC or RAS/mitogen-activated protein kinase syndromes related to BRAF variant. CFC syndrome, especially caused by BRAF variant, should be included in the differential diagnosis of patients with developmental and epileptic encephalopathies and hyperekplexia. Furthermore, we need to keep in mind that missense variants or the deletion of Leucine-485 may be associated with severe symptoms.

A novel mutation in the proteolytic domain of LONP1 causes atypical CODAS syndrome.

Cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in LONP1. It is characterized by intellectual disability, cataracts, delayed tooth eruption, malformed auricles and skeletal abnormalities. We performed whole-exome sequencing on a 12-year-old Japanese male with severe intellectual disability, congenital bilateral cataracts, spasticity, hypotonia with motor regression and progressive cerebellar atrophy with hyperintensity of the cerebellar cortex on T2-weighted images. We detected compound heterozygous mutation in LONP1. One allele contained a paternally inherited frameshift mutation (p.Ser100Glnfs*46). The other allele contained a maternally inherited missense mutation (p.Arg786Trp), which was predicted to be pathogenic by web-based prediction tools. The two mutations were not found in Exome Variant Server or our 575 in-house control exomes. Some features were not consistent with CODAS syndrome but overlapped with Marinesco-Sjögren syndrome, a multisystem disorder caused by a mutation in SIL1. An atypical mutation site may result in atypical presentation of the LONP1 mutation.

Patchy white matter hyperintensity in ring chromosome 18 syndrome.

Ring chromosome 18 syndrome is a chromosomal abnormality in which partial deletions occur at both ends of chromosome 18, that is, distally on the short and long arms. Previously reported brain magnetic resonance imaging (MRI) abnormalities include diffuse hyperintensity in the white matter, which has been regarded as hypomyelination because the gene for myelin basic protein production is located on the long arm of chromosome 18. We report the case of a 14-year-old boy with ring chromosome 18 syndrome, whose MRI showed patchy asymmetrical T2 and fluid-attenuated inversion-recovery hyperintensities in the deep white matter as well as diffuse hypomyelination. These patchy lesions may indicate demyelination or gliosis rather than hypomyelination. This result differs from previous reports.

The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent Polymerase.

A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H)-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H-Cu(II)-H). In this study, we investigated the effects of a metal cofactor, Mg(II) ion, on the TdT-catalyzed polymerization of H nucleotides. At a high Mg(II) concentration (10 mM), the reaction was halted after several H nucleotides were appended. In contrast, at lower Mg(II) concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the Mg(II) concentration. In the presence of excess Mg(II) ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with Mg(II) ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.

Bifacial Base-Pairing Behaviors of 5-Hydroxyuracil DNA Bases through Hydrogen Bonding and Metal Coordination.

A novel bifacial ligand-bearing nucleobase, 5-hydroxyuracil (U(OH) ), which forms both a hydrogen-bonded base pair (U(OH) -A) and a metal-mediated base pair (U(OH) -M-U(OH) ) has been developed. The U(OH) -M-U(OH) base pairs were quantitatively formed in the presence of lanthanide ions such as Gd(III) when U(OH) -U(OH) pairs were consecutively incorporated into DNA duplexes. This result established metal-assisted duplex stabilization as well as DNA-templated assembly of lanthanide ions. Notably, a duplex possessing U(OH) -A base pairs was destabilized by addition of Gd(III) ions. This observation suggests that the hybridization behaviors of the U(OH) -containing DNA strands are altered by metal complexation. Thus, the U(OH) nucleobase with a bifacial base-pairing property holds great promise as a component for metal-responsive DNA materials.

Highly specific unnatural base pair systems as a third base pair for PCR amplification.

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.

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.

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.

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.