Low-Noise Solid-State Nanopore Enhancing Direct Label-Free Analysis for Small Dimensional Assemblies Induced by Specific Molecular Binding.

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.

Rolling-Circle Replication in Mitochondrial DNA Inheritance: Scientific Evidence and Significance from Yeast to Human Cells.

Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance, have demonstrated that a rolling-circle replication mode exists in yeasts and human cells. In yeast, rolling-circle mtDNA replication mediated by homologous recombination is the predominant pathway for replication of wild-type mtDNA. In human cells, reactive oxygen species (ROS) induce rolling-circle replication to produce concatemers, linear tandem multimers linked by head-to-tail unit-sized mtDNA that promote restoration of homoplasmy from heteroplasmy. The event occurs ahead of mtDNA replication mechanisms observed in mammalian cells, especially under higher ROS load, as newly synthesized mtDNA is concatemeric in hydrogen peroxide-treated human cells. Rolling-circle replication holds promise for treatment of mtDNA heteroplasmy-attributed diseases, which are regarded as incurable. This review highlights the potential therapeutic value of rolling-circle mtDNA replication.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

Fluorometric determination of ssDNA based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

A method is described for the determination of DNA via nucleic acid amplification by using nucleic acid concatemers that result from DNA supersandwich self-assemblies (SSAs). The method employs two auxiliary probes to form self-assembled biotin SSAs. These exhibit strong fluorescence if labeled with intercalator SYBR Green I. In the presence of the target (as exemplified for a 30-mer), streptavidin is released from the surface of the functionalized magnetic microparticles (FMMPs) by competitive hybridization on the surface. However, the SSA products do not conjugate to the FMMPs. This leads to a large amount of SYBR Green I intercalated into the concatemers and eventually results in amplified fluorescence in the supernate. The SSA products can be prepared beforehand, and amplification therefore can be completed within 50 min. The method is more efficient than any other conventional amplification. The detection limit for the 30-mer is 26.4 fM which is better by a factor of 10 compared to other amplification methods. Conceivably, the method can be further extended to the determination of a wide variety of targets simply by replacing the sequences of the probes. Finally, this rapid and highly sensitive method was employed for detection of Ebola virus gene (≈30-mer) and ATP in spiked serum with satisfactory results. Graphical abstract A high sensitivity and efficiency bioassay is described based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

DNA concatemer-silver nanoparticles as a signal probe for electrochemical prostate-specific antigen detection.

In this study, we report a metallobioassay for ultrasensitive electrochemical detection of prostate-specific antigen (PSA) based on DNA hybridization chain reaction (HCR) for amplifying the signal, which is derived from silver nanoparticles (Ag NPs) on DNA concatemers. The assay mainly consists of primary antibody (Ab1), secondary antibody (Ab2) with primer, and a signal probe. In the presence of PSA, a sandwich structure with DNA concatemers was formed, and numerous Ag NPs were loaded on the DNA concatemers, resulting in a strong signal, which appeared within the applied potential (-0.2 V to 0.3 V) in the phosphate-buffered saline (PBS). Differential pulse voltammetry (DPV) was employed to evaluate the analytical performance. Under optimal conditions, the DPV peak current of Ag NPs at about +0.09 V (vs. SCE) increased linearly as the logarithm of PSA concentration increased from 0.1 pg mL-1 to 75 ng mL-1, and the detection limit of PSA was estimated to be 0.033 pg mL-1 at the signal to noise ratio of 3. In addition, the assay was evaluated with human serum samples, and satisfying results were obtained, indicating that the assay can achieve PSA detection in the serum sample.

A DNAzyme-powered cross-catalytic circuit for amplified intracellular imaging.

A facile cross-catalytic circuit is engineered for intracellular microRNA (miRNA) imaging by facilely integrating a catalytic hairpin assembly (CHA) amplifier and DNAzyme biocatalyst through an ingenious feedback loop. These two indispensable catalytic reactions play vital roles in executing high-performance signal amplification, as demonstrated experimentally and theoretically.

Analysis of the dominant mutation N188T of human connexin46 (hCx46) using concatenation and molecular dynamics simulation.

Connexins (Cx) are proteins that form cell-to-cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co-expressed wild-type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt-hCx46N188T and hCx46N188T-hCx46wt to analyze how hCx46N188T affected co-expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt-hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt-hCx46wt, while the hCx46N188T-hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co-expressing the variants.

Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

A phylogenomic approach to reconstruct interrelationships of main clupeocephalan lineages with a critical discussion of morphological apomorphies.

BACKGROUND: Previous molecular studies on the phylogeny and classification of clupeocephalan fishes revealed numerous new taxonomic entities. For re-analysing these taxa, we perform target gene capturing and subsequent next generation sequencing of putative ortholog exons of major clupeocephalan lineages. Sequence information for the RNA bait design was derived from publicly available genomes of bony fishes. Newly acquired sequence data comprising > 800 exon sequences was subsequently used for phylogenetic reconstructions. RESULTS: Our results support monophyletic Otomorpha comprising Alepocephaliformes. Within Ostariophysi, Gonorynchiformes are sister to a clade comprising Cypriniformes, Characiformes, Siluriformes and Gymnotiformes, where the interrelationships of Characiformes, Siluriformes and Gymnotiformes remain enigmatic. Euteleosts comprise four major clades: Lepidogalaxiiformes, Protacanthopterygii, Stomiatii, and Galaxiiformes plus Neoteleostei. The monotypic Lepidogalaxiiformes form the sister-group to all remaining euteleosts. Protacanthopterygii, comprising Argentini-, Esoci- and Salmoniformes, is sister to Stomiatii (Osmeriformes and Stomiatiformes) and Galaxiiformes plus Neoteleostei. CONCLUSIONS: Several proposed monophyla defined by morphological apomorphies within the Clupeocephalan phylogeny are confirmed by the phylogenetic estimates presented herein. However, other morphologically described groups cannot be reconciled with molecular phylogenies. Thus, numerous morphological apomoprhies of supposed monophyla are called into question. The interpretation of suggested morphological synapomorphies of otomorph fishes is strongly affected by the inclusion of deep-sea inhabiting, and to that effect morphologically adapted Alepocephaliformes. Our revision of these potential synapomorphies, in the context that Alepocephaliformes are otomorph fishes, reveals that only a single character of the total nine characters proposed as synapomorphic for the group is clearly valid for all otomorphs. Three further characters remain possible apomorphies since their status remains unclear in the deep-sea adapted Alepocephaliformes showing developmental lag and lacking a swim bladder. Further, our analysis places Galaxiiformes as sister group to neoteleosts, which contradicts some previous molecular phylogenetic studies. This needs further investigation from a morphological perspective, as suggested synapomophies for several euteleostean lineages are challenged or still lacking. For the verification of results presented herein, a denser phylogenomic-level taxon sampling should be applied.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.