Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

The terminal deoxynucleotidyl transferase (TdT) belongs to the X family of DNA polymerases. This unusual polymerase catalyzes the template-independent addition of random nucleotides on 3′-overhangs during V(D)J recombination. The biological function and intrinsic biochemical properties of the TdT have spurred the development of numerous oligonucleotide-based tools and methods, especially if combined with modified nucleoside triphosphates. Herein, we summarize the different applications stemming from the incorporation of modified nucleotides by the TdT. The structural, mechanistic, and biochemical properties of this polymerase are also discussed.     

DNA Nanoarchitectures: Steps towards Biological Applications

DNA's remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During recent decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile‐based assembly process is a useful tool to construct large and simple structures; the DNA origami method is suitable for the production of smaller, more sophisticated and well‐defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures in molecular and cellular biology.     

Salt-Toggled Capture Selection of Uric Acid Binding Aptamers

Uric acid is the end-product of purine metabolism in humans and an important biomarker for many diseases. To achieve the detection of uric acid without using enzymes, we previously selected a DNA aptamer for uric acid with a K_d of 1 μM but the aptamer required multiple Na⁺ ions for binding. Saturated binding was achieved with around 700 mM Na⁺ and the binding at the physiological condition was much weaker. In this work, a new selection was performed by alternating Mg²⁺-containing buffers with Na⁺ and Li⁺. After 13 rounds of selection, a new aptamer sequence named UA-Mg-1 was obtained. Isothermal titration calorimetry confirmed aptamer binding in both selection buffers, and the K_d was around 8 μM. The binding of UA-Mg-1 to UA required only Mg²⁺. This is an indicator of successful switching of metal dependency via the salt-toggled selection method. The UA-Mg-1 aptamer was engineered into a fluorescent biosensor based on the strand-displacement assay with a limit of detection of 0.5 μM uric acid in the selection buffer. Finally, comparison with the previously reported Na⁺-dependent aptamer and a xanthine/uric acid riboswitch was also made.     

The Three S's for Aptamer‐Mediated Control of DNA Nanostructure Dynamics: Shape,Self‐Complementarity,and Spatial Flexibility

DNA aptamers are ideal tools to enable modular control of the dynamics of DNA nanostructures. For molecular recognition, they have a particular advantage over antibodies in that they can be integrated into DNA nanostructures in a bespoke manner by base pairing or nucleotide extension without any complex bioconjugation strategy. Such simplicity will be critical upon considering advanced therapeutic and diagnostic applications of DNA nanostructures. However, optimizing DNA aptamers for functional control of the dynamics of DNA nanostructure can be challenging. Herein, we present three considerations—shape, self‐complementarity, and spatial flexibility—that should be paramount upon optimizing aptamer functionality. These lessons, learnt from the growing number of aptamer–nanostructure reports thus far, will be helpful for future studies in which aptamers are used to control the dynamics of nucleic acid nanostructures.     

Targeted Heating of Enzyme Systems Based on Photothermal Materials

This study demonstrates that the enzymatic reaction rate can be increased significantly by targeted heating of the microenvironment around the enzyme, while maintaining the reaction system at environmental temperature. Enzyme molecules are covalently attached to the surface of Fe₃O₄@reduced graphite oxide (rGO). Under visible-light irradiation, the reaction rate catalyzed by the enzyme–Fe₃O₄@rGO system is clearly enhanced relative to that of the free enzyme and a mixture of free enzyme and Fe₃O₄@rGO. This local heating mechanism contributes to promotion of the enzymatic reactions of the targeted heating of the enzyme (THE) system, which has been validated by using different enzymes, including lipase, glucose oxidase, and organophosphorus hydrolase. These results indicate that targeted heating of the catalytic centers has the same effect on speeding up reactions as that of traditional heating methods, which treat the whole reaction system. As an example, it is shown that the THE system promotes the sensitivity of an enzyme screen-printed electrode by 14 times at room temperature, which implies that the THE system can be advantageous in improving enzyme efficiency, especially if heating the entire system is impossible or could lead to degradation of substrates or damage of components, such as in vitro bioanalysis of frangible molecules or in vivo diagnosis.     

Amplified telomerase analysis by using rotating magnetic particles: the rapid and sensitive detection of cancer cells

A highly sensitive telomerase detection method that involves amplified telomerase analysis and the use of rotating magnetic particles has been developed. Magnetic particles, functionalized with a primer (1) that is recognized by telomerase, are mixed with a nucleotide mixture that includes biotinylated-dUTP, and telomerase-induced elongation of the primers proceeds with simultaneous biotin incorporation. Avidin-Horseradish peroxidase conjugate, coupled to biotin labels, yields the biocatalytic functional particles. Mixing the resulting particles with naphthoquinone-modified magnetic particles enables the optoelectronic detection of telomerase. Attraction of the magnetic particles to an electrode, followed by rotation of the particles, causes the electrocatalytic reduction of O(2) to H(2)O(2) and HRP-catalyzed oxidation of luminol (3); this results in chemilumunescence. The intensity of the emitted light depends on the telomerase content of the sample and the rotation speed of the particles. A minimum number of 10 cancer cells could be detected.     

Isolation and Characterization of RNA Aptamers against a Proteasome‐Associated Deubiquitylating Enzyme UCH37

Deubiquitylating (DUB) enzymes antagonize ubiquitin‐dependent protein degradation both before and after the substrates are engaged with proteasomes. UCH37 is one of three proteasome‐associated DUB enzymes in mammals and the only protease among them from the ubiquitin carboxyl‐terminal hydrolase (UCH) family. Here, we report the identification of specific RNA aptamers for UCH37 through in vitro selection, and we describe their inhibitory effects on the DUB activity of UCH37. The RNA aptamers significantly delayed RPN13‐mediated UCH37 activation and lowered total DUB activity of proteasomes, as measured by the hydrolysis of ubiquitin‐rhodamine 110. In addition, the UCH37 aptamers efficiently facilitated the hydrolysis of peptide‐based reporter substrates of proteasomes. Thus, the UCH37 aptamers might offer a possible strategy for removing toxic cellular proteins through enhancing proteasome activity.     

Recent Advances of DNA Tetrahedra for Therapeutic Delivery and Biosensing

The chemical and self-assembly properties of nucleic acids make them ideal for the construction of discrete structures and stimuli-responsive devices for a diverse array of applications. Amongst the various three-dimensional assemblies, DNA tetrahedra are of particular interest, as these structures have been shown to be readily taken up by the cell, by the process of caveolin-mediated endocytosis, without the need for transfection agents. Moreover, these structures can be readily modified with a diverse range of pendant groups to confer greater functionality. This minireview highlights recent advances related to applications of this interesting DNA structure including the delivery of therapeutic agents ranging from small molecules to oligonucleotides in addition to its use for sensing and imaging various species within the cell.     

Construction of T‐Motif‐Based DNA Nanostructures through Enzymatic Reactions

The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced through enzymatic reactions, they could be applied in various environments, including in vivo. Herein, an enzymatic method developed to construct a DNA nanostructure from a simple motif called a T‐motif is reported. A long, repeated structure was replicated from a circular template by rolling circle amplification and then cleaved into T‐motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder‐like nanostructure without purification or controlled annealing. This approach is widely applicable to constructing a variety of DNA nanostructures through enzymatic reactions.     

Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.     

膜在生物技术中的应用

综述了膜在生物技术方面的应用。概述了纳滤膜的基本原理。着重介绍了纳滤膜在食品工业，生物化工与制药工业，染料工业等方面的一些应用。     

Applications of DNA-Functionalized Proteins

As an important component that constitutes all the cells and tissues of the human body, protein is involved in most of the biological processes. Inspired by natural protein systems, considerable efforts covering many discipline fields were made to design artificial protein assemblies and put them into application in recent decades. The rapid development of structural DNA nanotechnology offers significant means for protein assemblies and promotes their application. Owing to the programmability, addressability and accurate recognition ability of DNA, many protein assemblies with unprecedented structures and improved functions have been successfully fabricated, consequently creating many brand-new researching fields. In this review, we briefly introduced the DNA-based protein assemblies, and highlighted the limitations in application process and corresponding strategies in four aspects, including biological catalysis, protein detection, biomedicine treatment and other applications.     

Glutathione Conjugation and DANN Adduct Formation of Diol Epoxides in V79 Cells Expressing Human Glutathione Transferase P1-1

V79 cells and cells over-expressing glutathione transferase (GST) P1-1 have been incubated with the (+)- and (?)-anti?enantiomers of trans?7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and trans?9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydrobenzo[c]chrysene (BCDE) and the formation of glutathione (GSH) conjugates and DNA adducts monitored. With (+)-anti?BPDE and (?)-anti?BCDE, the results demonstrate a several fold increase in conjugate formation concomitant with reduced levels of DNA adducts in GST expressing cells relative to control cells. Thus, the effects were restricted to the enantiomers with R?absolute configuration at the benzylic oxirane carbon. The rate of conjugate formation of BCDE relative to BPDE is significantly lower indicating reduced accessibility of the more lipophilic BCDE for GSTP1-1.     

Parallel analysis of two analytes in solutions or on surfaces by using a bifunctional aptamer: applications for biosensing and logic gate operations

A bifunctional aptamer that includes two aptamer units for cocaine and adenosine 5'-monophosphate (AMP) is blocked by a nucleic acid to form a hybrid structure with two duplex regions. The blocked bifunctional aptamer assembly is used as a functional structure for the simultaneous sensing of cocaine or AMP. The blocked bifunctional aptamer is dissociated by either of the two analytes, and the readout of the separation of the sensing structure is accomplished by a colorimetric detection, by a released DNAzyme, or by electronic means that use Faradaic impedance spectroscopy or field-effect transistors. In one configuration, the blocked bifunctional aptamer structure is separated by the substrates cocaine or AMP, and the displaced blocker units act as a horseradish peroxidase-mimicking DNAzyme that permits the colorimetric detection of the analytes. In the second system, the blocked bifunctional aptamer hybrid is associated with a Au electrode. The displacement of the aptamer by any of the substrates alters the interfacial electron transfer resistance at the electrode surface, thus providing an electronic signal for the sensing process. In the third configuration, the blocked aptamer hybrid is linked to the gate of a field-effect transistor device. The separation of the complex by means of any of the analytes, cocaine, or AMP alters the gate potential, and this allows the electronic transduction of the sensing process by following the changes in the gate-to-source potentials. The different systems enable not only the simultaneous detection of the two analytes, but they provide a functional assembly that performs a logic gate "OR" operation.     

Effects of Methoxyfenozide-Loaded Fluorescent Mesoporous Silica Nanoparticles on Plutella xylostella (L.) (Lepidoptera: Plutellidae) Mortality and Detoxification Enzyme Levels Activities

The diamond back moth, Plutella xylostella, causes severe damage at all crop stages, beside its rising resistance to all insecticides. The objective of this study was to look for a new control strategy such as application of insecticide-loaded carbon dot-embedded fluorescent mesoporous silica nanoparticles (FL-SiO₂ NPs). Two different-sized methoxyfenozide-loaded nanoparticles (Me@FL-SiO₂ NPs-70 nm, Me@FL-SiO₂ NPs-150 nm) were prepared, with loading content 15% and 16%. Methoxyfenozide was released constantly from Me@FL-SiO₂ NPs only at specific optimum pH 7.5. The release of methoxyfenozide from Me@FL-SiO₂ NPs was not observed other than this optimum pH, and therefore, we checked and controlled a single release condition to look out for the different particle sizes of insecticide-loaded NPs. This pH-responsive release pattern can find potential application in sustainable plant protection. Moreover, the lethal concentration of the LC₅₀ value was 24 mg/L for methoxyfenozide (TC), 14 mg/L for Me@FL-SiO₂ NPs-70 nm, and 15 mg/L for Me@FL-SiO₂ NPs-150 nm after 72 h exposure, respectively. After calculating the LC₅₀, the results predicted that Me@FL-SiO₂ NPs-70 nm and Me@FL-SiO₂ NPs-150 nm exhibited better insecticidal activity against P. xylostella than methoxyfenozide under the same concentrations of active ingredient applied. Moreover, the activities of detoxification enzymes of P. xylostella were suppressed by treatment with insecticide-loaded NPs, which showed that NPs could also be involved in reduction of enzymes. Furthermore, the entering of FL-SiO₂ NPs into the midgut of P. xylostella was confirmed by confocal laser scanning microscope (CLSM). For comparison, P. xylostella under treatment with water as control was also observed under CLSM. The control exhibited no fluorescent signal, while the larvae treated with FL-SiO₂ NPs showed strong fluorescence under a laser excitation wavelength of 448 nm. The reduced enzyme activities as well as higher cuticular penetration in insects indicate that the nano-based delivery system of insecticide could be potentially applied in insecticide resistance management.     

纳米技术在聚氨酯工业中的应用

简要介绍了聚氨酯材料的优缺点,并综述了纳米技术在聚氨酯涂料、胶粘剂、塑料以及聚氨酯树脂基复合材料中的应用现状。     

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2⁺ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2⁺ cells in a background of HER2⁻ cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.     

The N‐Terminal Domain of the Pullulanase from Anoxybacillus sp. WB42 Modulates Enzyme Specificity and Thermostability

Anoxybacillus sp. WB42 pullulanase (PulWB42) is a novel thermophilic amylopullulanase that was assigned to the glycoside hydrolase family 13 subfamily 14 (GH13_14) type I pullulanases in the carbohydrate‐active enzymes database. Its N‐terminal domain (Met1–Phe101) was identified as the carbohydrate‐binding module 68 (CBM68) by homology modeling. The N‐domain‐deleted PulWB42 exhibited an equivalent Michaelis constant (K_m) for pullulan and significant decreases in pullulytic activity, amylose selectivity, and thermostability relative to PulWB42 having a high α‐amylase‐to‐pullulanase activity ratio. Furthermore, the replacement of Ala90 or Arg93 significantly changed the substrate specificity and catalytic efficiency of PulWB42, whereas Q87A, L173D, and H5A/R6A/T7A showed improvements in thermostability and changes in catalytic kinetics. Therefore, the N domain of PulWB42 is not essential for catalysis, but it does modulate enzyme catalysis, especially with respect to substrate specificity. The modulation was achieved mainly by the Leu86–Arg93 segment adjacent to the CBM48 domain and the catalytic A domain in the modeled structure of PulWB42.     

An RNA Aptamer That Selectively Inhibits the Enzymatic Activity of Protein Tyrosine Phosphatase 1B in vitro

SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a K_i of less than 600 pM . This slow‐binding, high‐affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC‐PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single‐nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide‐based compounds in combating several human cancers and metabolic diseases.     

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.