Imaging mRNA in vitro and in vivo with nanofirecracker probes via intramolecular hybridization chain reaction.

Hybridization chain reaction (HCR) based enzyme-free amplification techniques have recently been developed for the visualization of intracellular messenger RNA (mRNA). However, the slow kinetics and potential interference with the intricate biological environments hinder its application in the clinic and in vivo. Herein, we designed a nanofirecracker probe-based strategy using intramolecular hybridization chain reaction (IHCR) amplifier for rapid, efficient, sensitive, specific detection and imaging of survivin mRNA both in vitro and vivo. Two probes, HP1 and HP2, in IHCR were simultaneously incorporated into a DNA nanowire scaffolds to bring HP1 and HP2 to close proximity on the assembled nanowire scaffolds. Empowered by the DNA nanowire scaffolds and spatial confinement effect, the nanofirecracker probe-based IHCR sensing system exhibited improved biostability, accelerated reaction kinetics, and enhanced signal amplification. This new strategy has been successfully applied to imaging mRNA in both cultured cells and in mice. Importantly, this novel sensing method was capable of detecting survivin mRNA in clinical blood samples from subjects with colorectal cancer. Thus, this novel nanofirecracker probe-based IHCR strategy holds great potential in advancing both biomedical research and in molecular diagnostics.

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis.

Structural diverse natural products like ribosomally synthesized and posttranslationally modified peptides (RiPPs) display a wide range of biological activities. Currently, the mechanism of an uncommon reaction step during the biosynthesis of 3-thiaglutamate (3-thiaGlu) is poorly understood. The removal of the ß-carbon from the Cys in the TglA-Cys peptide catalyzed by the TglHI holoenzyme remains elusive. Here, we present three crystal structures of TglHI complexes with and without bound iron, which reveal that the catalytic pocket is formed by the interaction of TglH-TglI and that its activation is conformation dependent. Biochemical assays suggest a minimum of two iron ions in the active cluster, and we identify the position of a third iron site. Collectively, our study offers insights into the activation and catalysis mechanisms of the non-heme dioxygen-dependent holoenzyme TglHI. Additionally, it highlights the evolutionary and structural conservation in the DUF692 family of biosynthetic enzymes that produce diverse RiPPs.

Spatial confinement-based Figure-of-Eight nanoknots accelerated simultaneous detection and imaging of intracellular microRNAs.

Developing highly efficient and reliable methods for simultaneous imaging of microRNAs in living cells is often appealed to understanding their synergistic functions and guiding the diagnosis and treatment of human diseases, such as cancers. In this work, we rationally engineered a four-arm shaped nanoprobe that can be stimuli-responsively tied into a Figure-of-Eight nanoknot via spatial confinement-based dual-catalytic hairpin assembly (SPACIAL-CHA) reaction and applied for accelerated simultaneous detection and imaging of different miRNAs in living cells. The four-arm nanoprobe was facilely assembled from a cross-shaped DNA scaffold and two pairs of CHA hairpin probes (21HP-a and 21HP-b for miR-21, while 155HP-a and 155HP-b for miR-155) via the "one-pot" annealing method. The DNA scaffold structurally provided a well-known spatial-confinement effect to improve the localized concentration of CHA probes and shorten their physical distance, resulting in an enhanced intramolecular collision probability and accelerating the enzyme-free reaction. The miRNA-mediated strand displacement reactions can rapidly tie numerous four-arm nanoprobes into Figure-of-Eight nanoknots, yielding remarkably dual-channel fluorescence proportional to the different miRNA expression levels. Moreover, benefiting from the nuclease-resistant DNA structure based on the unique arched DNA protrusions makes the system ideal for operating in complicated intracellular environments. We have demonstrated that the four-arm-shaped nanoprobe is superior to the common catalytic hairpin assembly (COM-CHA) in stability, reaction speed, and amplification sensitivity in vitro and living cells. Final applications in cell imaging have also revealed the capacity of the proposed system for reliable identification of cancer cells (e.g., HeLa and MCF-7) from normal cells. The four-arm nanoprobe shows great potential in molecular biology and biomedical imaging with the above advantages.

Branch-Shaped Trapping Device Regulates Accelerated Catalyzed Hairpin Assembly and Its Application for MicroRNA In Situ Imaging.

Enzyme-free DNA strand displacement process is often practical when detecting miRNAs expressed at low levels in living cells. However, the poor kinetics, tedious reaction period, and multicomponent system hamper its in vivo applications to a great extent. Herein, we design a branch-shaped trapping device (BTD)-based spatial confinement reactor and applied it for accelerated miRNA in situ imaging. The reactor consists of a pair of trapped probe-based catalyzed hairpin assembly (T-CHA) reactions attached around the BTD. The trapping device naturally offered CHA reactions a good spatial-confinement effect by integrating the metastable probes (MHPa and MHPb) of the traditional CHA with the four-branched arm of BTD, which greatly improved the localized concentration of probes and shortened their physical distance. The autonomous and progressive walk of miRNA on the four-arm nanoprobes via T-CHA can rapidly tie numerous four-arm nanoprobes into figure-of-eight nanoknots (FENs), yielding strong fluorescence that is proportional to the miRNA expression level. The unique nanoarchitecture of the FEN also benefits the restricted freedom of movement (FOM) in a confined cellular environment, which makes the system ideally suitable for in situ imaging of intracellular miRNAs. In vitro and in situ analyses also demonstrated that the T-CHA overall outperformed the dissociative probe-based CHA (D-CHA) in stability, reaction speed, and amplification sensitivity. The final application of the T-CHA-based four-arm nanoprobe for imagings of both cancer cells and normal cells shows the potential of the platform for accurately and timely revealing miRNA in biological systems.

In situ imaging miRNAs using multifunctional linear DNA nanostructure.

The microRNAs (miRNAs) play a critical role in many biological processes and are essential biomarkers for diagnosing disease. However, the sensitive and specific quantification of microRNAs (miRNAs) expression in living cells still faces a huge challenge. Our study designed a multifunctional linear DNA nanostructure (MLN) as a carrier of molecular beacons (MB-21) for detecting and intracellular imaging miRNA-21. The MLN-MB consists of three parts: aptamer, MLN, and MB-21. The aptamer (AS1411) could media MLN-MB enter live cells without additional transfection reagents. Once inside the cells, the intracellular miRNA-21 could hybridize the MB-21s, resulting in significantly enhanced fluorescence signals. The whole process was enzyme-free, autonomous, and continuous, which avoided the necessity of adding external fuel strands or enzymes. We demonstrated that the MLN-MB could be used to screen the miRNA-21 with a detection limit of 320 pM in a short time (10 min) and show high specificity toward miRNA-21 against other miRNAs. Moreover, the proposed MLN-MB could detect the miRNA-21 in complex matrixes stably. With its outstanding stability, dual recognition, and biocompatibility, MLN-MB is capable of delivering into living cells to identify specific cancer cells. Therefore, our sensing approach, with high sensitivity, specificity, and simplicity advantages, holds great potential for early cancer diagnosis.

Highly sensitive detection and intracellular imaging of MicroRNAs based on target-triggered cascade catalytic hairpin assembly.

MicroRNAs (miRNAs) have been identified as important biomarkers with great significance for diagnosis and treatment of various diseases. However, their unique properties, such as small size, high sequence homology, and low abundance, make quantitative analysis of miRNAs extremely challenging. Herein, we reported a cascade catalytic hairpin assembly (CCHA) for sensitive and selective detection of miRNA with three kinds of hairpin probes (HP1, HP2, and HP3). In the presence of target miRNA, a series of toehold-mediated intermolecular DNA strand displacement and hybridization was activated among HP1, HP2, and HP3 to assembly numbers of DNA nanoobjects. During this period, the fluorescence response was greatly intensified to indicate the presence and expression level of interested target miRNA. We have demonstrated that the proposed method exhibits a high assay sensitivity to detect low concentration target and an excellent sequence specificity to distinguish even a single-nucleotide difference in vitro. Moreover, we also demonstrated that our design enables the intracellular imaging of miRNA in live cancer and normal cells. These results showing the promising potential of our CCHA for powerful biosensing, clinic diagnosis, or prognosis.

Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis.

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB-MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes.

Harnessing anti-tumor and tumor-tropism functions of macrophages via nanotechnology for tumor immunotherapy.

Reprogramming the immunosuppressive tumor microenvironment by modulating macrophages holds great promise in tumor immunotherapy. As a class of professional phagocytes and antigen-presenting cells in the innate immune system, macrophages can not only directly engulf and clear tumor cells, but also play roles in presenting tumor-specific antigen to initiate adaptive immunity. However, the tumor-associated macrophages (TAMs) usually display tumor-supportive M2 phenotype rather than anti-tumor M1 phenotype. They can support tumor cells to escape immunological surveillance, aggravate tumor progression, and impede tumor-specific T cell immunity. Although many TAMs-modulating agents have shown great success in therapy of multiple tumors, they face enormous challenges including poor tumor accumulation and off-target side effects. An alternative solution is the use of advanced nanostructures, which not only can deliver TAMs-modulating agents to augment therapeutic efficacy, but also can directly serve as modulators of TAMs. Another important strategy is the exploitation of macrophages and macrophage-derived components as tumor-targeting delivery vehicles. Herein, we summarize the recent advances in targeting and engineering macrophages for tumor immunotherapy, including (1) direct and indirect effects of macrophages on the augmentation of immunotherapy and (2) strategies for engineering macrophage-based drug carriers. The existing perspectives and challenges of macrophage-based tumor immunotherapies are also highlighted.

Overcoming Radioresistance in Tumor Therapy by Alleviating Hypoxia and Using the HIF-1 Inhibitor.

Radiotherapy has been extensively used to treat cancer patients because it can effectively damage most solid tumors without penetration limits. A hypoxic microenvironment in solid tumors leads to severe radioresistance and expression of hypoxic inducible factor-1 (HIF-1), which results in poor efficacy of radiotherapy alone. Herein, we report the excellent efficacy of radiotherapy achieved using a new type of yolk-shell Cu2-xSe@PtSe (CSP) nanosensitizer functionalized with the HIF-1α inhibitor acriflavine (ACF). We prepare the CSP nanosensitizer through the interfacial redox reactions between chloroplatinic acid and Cu2-xSe nanoparticles (CS) and then functionalize the nanosensitizer with ACF through their electrostatic interactions. We show that the synthesized CSP nanosensitizer can arrest the cell cycle (i.e., at the gap 2/mitosis (G2/M) phases) of tumor cells to enhance their sensitivity to X-rays and decompose endogenous H2O2 into O2 to reduce hypoxia and increase the production of reactive oxygen species, which leads to severe damage to DNA double strands and apoptosis of tumor cells. We also show that the ACF on the surface of CSP nanoparticles can effectively reduce the expression of HIF-1α. All these effects lead to a low vascular endothelial growth factor, low density of microvessels in tumor, decreased cell proliferation, and increased cell apoptosis, which synergistically and drastically enhance the efficacy of radiotherapy. This work provides insights and guidance for developing novel nanosensitizers to enhance the efficacy of radiotherapy.

[Expression of hypoxia-inducible factor-1alpha, vascular endothelial growth factor and sFlt-1 in preeclampsia placenta].

OBJECTIVE: To investigate the expression and correlation of hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and sFlt-1 in the preeclampsia placenta, and discuss their significance in the pathogenesis of preeclampsia. METHODS: Placentas were collected from 20 pregnant women with preeclampsia as study group and 15 normal pregnant women as control group. The expressions of HIF-1alpha, VEGF and sFlt-1 protein were semi-quantitatively analyzed with immunohistochemical assay and mRNA level was determined using reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: (1) the expression of HIF-1alpha and sFlt-1 protein in preeclampsia group obviously increased. Strong (+++) positive expression was observed in 9 and 11 cases respectively, significantly higher than in control group (2 and 3 cases) (P < 0.05), however, VEGF expression obviously reduced in preeclampsia group (P < 0.01). (2) the level of HIF-1alpha and sFlt-1 mRNA in preeclamptic placenta was 0.604 +/- 0.013, 0.898 +/- 0.041, significantly higher than 0.208 +/- 0.007 and 0.559 +/- 0.244 in normal placenta (P < 0.05). Although the level of VEGF mRNA increased in preeclampsia placenta, it was not significantly different from that in normal placenta (P > 0.05). The ratio of VEGF mRNA/sFlt-1mRNA obviously reduced in preeclampsia group and was significantly lower than in control group (P < 0.05). (3) in preeclampsia group, HIF-1alpha mRNA expression was positively correlated with the expression of sFlt-1 mRNA (r = 0.577, P < 0.05), and negatively correlated with the ratio of VEGF mRNA/sFlt-1 mRNA (r = -0.376, P < 0.05). CONCLUSION: Abnormal high HIF-1alpha expression in preeclampsia placenta indicates that HIF-1alpha might play an important role in the pathogenesis of preeclampsia, possibly through affecting the cytotrophoblastic invasion and placental vascular reconstruction via the modulation of VEGF and sFlt-1 gene transcription.