Polymer-based chemical-nose systems for optical-pattern recognition of gut microbiota.

Gut-microbiota analysis has been recognized as crucial in health management and disease treatment. Metagenomics, a current standard examination method for the gut microbiome, is effective but requires both expertise and significant amounts of general resources. Here, we show highly accessible sensing systems based on the so-called chemical-nose strategy to transduce the characteristics of microbiota into fluorescence patterns. The fluorescence patterns, generated by twelve block copolymers with aggregation-induced emission (AIE) units, were analyzed using pattern-recognition algorithms, which identified 16 intestinal bacterial strains in a way that correlates with their genome-based taxonomic classification. Importantly, the chemical noses classified artificial models of obesity-associated gut microbiota, and further succeeded in detecting sleep disorder in mice through comparative analysis of normal and abnormal mouse gut microbiota. Our techniques thus allow analyzing complex bacterial samples far more quickly, simply, and inexpensively than common metagenome-based methods, which offers a powerful and complementary tool for the practical analysis of the gut microbiome.

Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

Characterization of an extracellular α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.

α-Xylosidases release the α-D-xylopyranosyl side chain from di- and oligosaccharides derived from xyloglucans and are involved in xyloglucan degradation. In this study, an extracellular α-xylosidase, named AxyB, is identified and characterized in Aspergillus oryzae. AxyB belongs to the glycoside hydrolase family 31 and releases D-xylose from isoprimeverose (α-D-xylopyranosyl-(1 → 6)-D-glucopyranose) and xyloglucan oligosaccharides. In the hydrolysis of xyloglucan oligosaccharides (XLLG, Glc4Xyl3Gal2 nonasaccharide; XLXG/XXLG, Glc4Xyl3Gal1 octasaccharide; and XXXG, Glc4Xyl3 heptasaccharide), AxyB releases one molecule of the xylopyranosyl side chain attached to the non-reducing end of the ß-1,4-glucan main chain of these xyloglucan oligosaccharides to yield GLLG (Glc4Xyl2Gal2), GLXG/GXLG (Glc4Xyl2Gal1), and GXXG (Glc4Xyl2). A. oryzae has both extracellular and intracellular α-xylosidase, suggesting that xyloglucan oligosaccharides are degraded by a combination of isoprimeverose-producing oligoxyloglucan hydrolase and intracellular α-xylosidase and a combination of extracellular α-xylosidase and ß-glucosidase(s) in A. oryzae. KEY POINTS: • An extracellular α-xylosidase, AxyB, is identified in Aspergillus oryzae. • AxyB releases the xylopyranosyl side chain from xyloglucan oligosaccharides. • Different sets of glycosidases degrade xyloglucan oligosaccharides in A. oryzae.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Quantitative analysis of global 5-methyl- and 5-hydroxymethylcytosine in TET1 expressed HEK293T cells.

DNA methylation at the 5-position of cytosine bases (5-methylcytosine, 5mC) in genomic DNA is representative epigenetic modification and is involved in many cellular processes, including gene expression and embryonic development. The hydroxylation of 5mC provide 5-hydroxymethylcytosine (5hmC), the so-called sixth base rediscovered recently in mammalian cells, is also considered to act as an epigenetic regulator. We report herein the immunochemical assessment of 5hmC achieved by an enzyme-linked immunosorbent assay (ELISA) using our linker technology. The keys to this assay are 1) the immobilization of genomic DNA with the bifunctional linker molecule, and 2) quantitative analysis by using guaranteed standard samples containing defined amounts of 5hmC. We succeeded in the sensitive and quantitative detection of 5hmC as well as 5mC in HEK293T cells transfected with TET1, and also monitored the effect of ascorbate on the TET1 catalyzed conversion of 5mC to 5hmC. Our linker technology enables the rapid and stable immobilization of genomic samples and thus contributes to the realization of a reproducible 5hmC evaluation method.

PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody-drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product-single-stranded DNA conjugated at the carboxyl terminus of VHH-retained its affinity for EGFR. To investigate whether this VHH-single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100-500 bp DNA. We confirmed the amplification of the VHH-double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

Development of gapmer antisense oligonucleotide with deoxyribonucleic guanidine (DNG) modifications.

The properties of gapmer antisense oligonucleotide (ASO) flanked by deoxyribonucleic guanidine (DNG) were investigated for the potential application in antisense technology. DNG is a unique nucleotide analog which has a positively charged internucleotide guanidinium linkage instead of negatively charged phosphodiester backbone linkage. We prepared a gapmer ASO containing DNG units at both wings of the sequence and compared its properties with 2',4'-BNA/LNA gapmer ASOs with phosphorothioate (PS) backbone. Although DNG gapmer showed no stabilizing effect on the duplex formation with target RNA, the DNG modification was found to be tolerant to exonuclease digestion. Furthermore, DNG gapmer can induce RNase H-mediated cleavage of target RNA molecule, a requisite property for the antisense strategy. Therefore, the DNG gapmer developed in this study could be an interesting and useful candidate for the development of potent ASOs.

Use of the kojA promoter, involved in kojic acid biosynthesis, for polyketide production in Aspergillus oryzae: implications for long-term production.

BACKGROUND: Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae. RESULTS: A kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days. CONCLUSIONS: The combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.

Sequential Assessment of Multiple Epigenetic Modifications of Cytosine in Whole Genomic DNA by Surface Plasmon Resonance.

Since the discovery of the active DNA demethylation pathway in mammals, numerous efforts have been made to distinguish epigenetic cytosine variants, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). However, the rapid discrimination of multiple cytosine variants in DNA remains challenging because the conventional assays require time-consuming DNA pretreatments, such as enzymatical digestion and chemical conversion. Here we demonstrated the high-throughput discrimination of four cytosine variants in DNA by using a sequential surface-plasmon-resonance (SPR)-based immunochemical assay. The target DNAs were biotinylated in one step with a bifunctional linker 1 and robustly immobilized on a streptavidin-coated sensor surface to hold them in place during an alkali washing designed to remove residual antibodies. By repeating the injection of antibodies and washing, we achieved a sequential assessment of cytosine variants in identical DNA and identified the yield of in vitro 5mC oxidation in genomic DNA by the ten-eleven translocation 1 (TET1) enzyme. These results demonstrated that our sequential SPR-based immunochemical assay was effective for evaluating multiple epigenetic modifications in a whole genome with a single row operation without time-consuming DNA pretreatments.

Immobilization of DNA on Biosensing Devices with Nitrogen Mustard-Modified Linkers.

Immobilization of DNA is an important step in relation to DNA-based biosensors and bioassays with multiple applications. This unit describes synthesis and applications of novel bifunctional linker molecules containing nitrogen mustard and one of two types of functional groups: cyclic disulfide or biotin. Two ways of immobilizing DNA on a surface are described. With the first method, a bifunctional alkylating linker molecule is first reacted with the target DNA to form alkylated DNA and then immobilized on a specific surface. With the second method, the bifunctional alkylating linker molecule is first attached to the surface, and then the target DNA is immobilized through an alkylating reaction with a nitrogen mustard moiety. We have also achieved immunochemical detection and quantification of 5-methylcytosine in a target DNA immobilized by the above methods. The methods for immobilization of intact DNA using novel bifunctional linker molecules are applicable to a wide range of biological analysis techniques. © 2019 by John Wiley & Sons, Inc.

Immobilization of DNA with nitrogen mustard-biotin conjugate for global epigenetic analysis.

We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.

Synthesis and Application of Interstrand Cross-Linked Duplexes by Covalently Linking a Pair of Abasic Sites.

Interstrand cross-linking of DNA or RNA inhibits the double strands from dissociating into single strands. This article contains detailed procedures for the synthesis of a novel interstrand cross-linker that comprises a bis-aminooxy naphthalene derivative and a description of its use in the preparation of sequence-specific interstrand cross-linked oligonucleotide duplexes. The interstrand cross-linker covalently connects a pair of apurinic/apyrimidinic sites in DNA/RNA duplexes with bis(aminooxy) groups. The resulting oxime linkages are stable under physiological conditions and greatly improve the thermal stability of the duplex. In addition, we construct a novel anti-miRNA oligonucleotide (AMO) flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs). AMO flanked by CLs at the 5'- and 3'-termini exhibited high inhibition activity toward miRNA function in cells. The novel interstrand cross-linker indicates potent activity and is applicable in biophysical studies, oligonucleotide therapeutics, and materials science. © 2018 by John Wiley & Sons, Inc.

An alkylating immobilization linker for immunochemical epigenetic assessment.

A bifunctional linker molecule containing nitrogen mustard and a cyclic disulfide group has been developed for the covalent immobilization of intact DNA, which allows quantitative analysis of epigenomic modification in immobilized DNA using SPR-based immune sensing.

Development of a 3'-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex.

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5'-termini of oligonucleotides; however, the ssH linker is not appropriate for 3'-terminal modification because it undergoes intramolecular trans-acylation under heat-aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat-alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3'-revH linker was efficiently linked with the 5'-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2'-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3'-terminal modification of oligonucleotides but also expands the utility range in combination with the 5'-ssH linker.

Electrochemical properties of interstrand cross-linked DNA duplexes labeled with Nile blue.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

Synthesis and application of highly reactive amino linkers for functional oligonucleotides.

Oligonucleotides are functionalized by conjugation with a variety of molecules, and aliphatic amino linkers have been frequently used as a tether for their modifications. This unit describes the syntheses and applications of novel amino linkers having a carbamate structure. Two major chemical properties of the primary amine are induced by the neighboring effect of the carbamate group, which are found to be optimum in an aminoethyl carbamate structure. First, the hydrophobic monomethoxytrityl group can be rapidly removed from the aminoethyl carbamate under very mild acidic conditions, while the deprotection is not completed in standard aliphatic amines even under high acid concentration. This significant feature enables the convenient purification of amino-modified oligonucleotides by using the hydrophobic interaction of the monomethoxytrityl group with a reverse-phase resin. Second, the introduction of the carbamate linkage reduces the pK(a) value of the neighboring primary amine, resulting in an increase in the conjugation yields with various functional molecules, such as those having active esters. The novel amino linkers that have an aminoethyl carbamate linkage indicate potent activity and are applicable for the preparation of various functional oligonucleotides.

Interstrand cross-link of DNA by covalently linking a pair of abasic sites.

A pair of apurinic/apyrimidinic sites formed in DNA has been covalently connected with bis(aminooxy) derivatives. The efficacy of the interstrand cross-link is associated with the structural tethering of two aminooxy groups. The interstrand cross-link constructed stable DNA scaffolds for enzyme alignment.

Construction of highly reactive probes for abasic site detection by introduction of an aromatic and a guanidine residue into an aminooxy group.

Abasic sites (AP sites) arise from hydrolysis of glycosidic bonds of DNA that is damaged by various external and internal processes; unrepaired AP sites give rise to genetic mutations. We have constructed highly reactive AP-site-detecting probes by introducing a hydrophobic and a hydrophilic residue in an aminooxy group. Synthesized probes containing either a naphthalene or a guanidine residue conjugate effectively with AP sites. In particular, a probe containing both functional groups shows the highest reaction rate, indicating that the hydrophobic and hydrophilic interactions act cooperatively in reaction with AP sites. The guanidine residue also contributes to the solubility of the molecules in aqueous media. The biotinylated probes provide much more sensitive detection of AP sites in genomic DNA than the conventional aldehyde-reactive probe.

Development of novel chemical probes to detect abasic sites in DNA.

We chemically synthesized a series of aminooxy derivatives to develop novel probes for sensitive detection of abasic (AP) sites in DNA. The results of the conjugation reactions showed that the probes could efficiently react to AP sites by introducing an aromatic or a guanidino group in their structures. In particular, the probe having both functional groups showed the most effective reactivity, indicating that hydrophobic and electrostatic interactions cooperatively acted in the reaction of the probe to AP sites. We then synthesized a biotinylated probe and succeeded in more sensitive detection of AP sites in genomic DNA than with the conventional aldehyde reactive probe (ARP).

Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5'-terminal modification.

Solid-support conjugation at the 5'-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation. In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a solid-support due to rapid removal of the amino-protecting group, and activated amino acids or cholesterol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers. Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conjugation of functional molecules.