Symmetric subgenomes and balanced homoeolog expression stabilize the establishment of allopolyploidy in cyprinid fish.

BACKGROUND: Interspecific postzygotic reproduction isolation results from large genetic divergence between the subgenomes of established hybrids. Polyploidization immediately after hybridization may reset patterns of homologous chromosome pairing and ameliorate deleterious genomic incompatibility between the subgenomes of distinct parental species in plants and animals. However, the observation that polyploidy is less common in vertebrates raises the question of which factors restrict its emergence. Here, we perform analyses of the genome, epigenome, and gene expression in the nascent allotetraploid lineage (2.95 Gb) derived from the intergeneric hybridization of female goldfish (Carassius auratus, 1.49 Gb) and male common carp (Cyprinus carpio, 1.42 Gb), to shed light on the changes leading to the stabilization of hybrids. RESULTS: We firstly identify the two subgenomes derived from the parental lineages of goldfish and common carp. We find variable unequal homoeologous recombination in somatic and germ cells of the intergeneric F1 and allotetraploid (F22 and F24) populations, reflecting high plasticity between the subgenomes, and rapidly varying copy numbers between the homoeolog genes. We also find dynamic changes in transposable elements accompanied by genome merger and duplication in the allotetraploid lineage. Finally, we observe the gradual decreases in cis-regulatory effects and increases in trans-regulatory effects along with the allotetraploidization, which contribute to increases in the symmetrical homoeologous expression in different tissues and developmental stages, especially in early embryogenesis. CONCLUSIONS: Our results reveal a series of changes in transposable elements, unequal homoeologous recombination, cis- and trans-regulations (e.g. DNA methylation), and homoeologous expression, suggesting their potential roles in mediating adaptive stabilization of regulatory systems of the nascent allotetraploid lineage. The symmetrical subgenomes and homoeologous expression provide a novel way of balancing genetic incompatibilities, providing a new insight into the early stages of allopolyploidization in vertebrate evolution.

Design and application of DNA nanostructures for organelle-targeted delivery of anticancer drugs.

INTRODUCTION: DNA nanostructures targeting organelles are of great significance for the early diagnosis and precise therapy of human cancers. This review is expected to promote the development of DNA nanostructure-based cancer treatment with organelle-level precision in the future. AREAS COVERED: In this review, we introduce the different principles for targeting organelles, summarize the progresses in the development of organelle-targeting DNA nanostructures, highlight their advantages and applications in disease treatment, and discuss current challenges and future prospects. EXPERT OPINION: Accurate targeting is a basic problem for effective cancer treatment. However, current DNA nanostructures cannot meet the actual needs. Targeting specific organelles is expected to further improve the therapeutic effect and overcome tumor cell resistance, thereby holding great practical significance for tumor treatment in the clinic. With the deepening of the research on the molecular mechanism of disease development, especially on tumorigenesis and tumor progression, and increasing understanding of the behavior of biological materials in living cells, more versatile DNA nanostructures will be constructed to target subcellular organelles for drug delivery, essentially promoting the early diagnosis of cancers, classification, precise therapy and the estimation of prognosis in the future.

Stimuli-Responsive Autonomous-Motion Molecular Machine for Sensitive Simultaneous Fluorescence Imaging of Intracellular MicroRNAs.

DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.

Programmably tiling rigidified DNA brick on gold nanoparticle as multi-functional shell for cancer-targeted delivery of siRNAs.

Small interfering RNA (siRNA) is an effective therapeutic to regulate the expression of target genes in vitro and in vivo. Constructing a siRNA delivery system with high serum stability, especially responsive to endogenous stimuli, remains technically challenging. Herein we develop anti-degradation Y-shaped backbone-rigidified triangular DNA bricks with sticky ends (sticky-YTDBs) and tile them onto a siRNA-packaged gold nanoparticle in a programmed fashion, forming a multi-functional three-dimensional (3D) DNA shell. After aptamers are arranged on the exterior surface, a biocompatible siRNA-encapsulated core/shell nanoparticle, siRNA/Ap-CS, is achieved. SiRNAs are internally encapsulated in a 3D DNA shell and are thus protected from enzymatic degradation by the outermost layer of YTDB. The siRNAs can be released by endogenous miRNA and execute gene silencing within tumor cells, causing cell apoptosis higher than Lipo3000/siRNA formulation. In vivo treatment shows that tumor growth is completely (100%) inhibited, demonstrating unique opportunities for next-generation anticancer-drug carriers for targeted cancer therapies.

Rigidified DNA Triangle-Protected Molecular Beacon from Endogenous Nuclease Digestion for Monitoring microRNA Expression in Living Cells.

Utilizing the nucleic acid-based self-assembly technology, Y-shaped backbone-rigidified DNA triangles with substantially enhanced nuclease resistance are built by designing a Y-shaped backbone in the center of a planar DNA triangle. Along this line, we developed aptamer-targeted DNA triangle-based molecular beacon (Apt-Tri-MB) probes for monitoring the microRNA expression in living cells with high sensitivity and specificity. For the Apt-Tri-MB probe, the MB is protected by the DNA triangle from unwanted enzymatic digestion, and a targeting ligand aptamer is introduced to endow the MB with active tumor cell-targeting capability. Thus, the digestion-induced false-positive signal is avoided, and the background fluorescence, which originates from the passive cell uptake (e.g., transfection) of reporting probes, is substantially suppressed. The imaging capability of the Apt-Tri-MB is superior to the commercial transfection agent-based counterpart and exhibits good universality suitable for imaging different miRNAs by changing the recognition fragment of the MB. Meanwhile, the disadvantages are efficiently circumvented, including the susceptibility of nucleic acids to nuclease-mediated degradation, inability of MB probes to enter cells, lipofectamine-determined cellular cytotoxicity, and nontargeting cell uptake. Inspired by the Y-shaped backbone-rigidified Apt-Tri-MB, we also constructed X-shaped backbone-rigidified quadrangle-based probes (Apt-Qua-MB). The experimental results show that cell imaging and antidegradation capability of Apt-Qua-MB are comparable with Apt-Tri-MB. As a proof-of-concept study, the Apt-Tri-MB is expected to open an exciting avenue for the further application of nucleic acid probes in the cellular level research and clinical disease diagnosis.

Oriented Tetrahedron-Mediated Protection of Catalytic DNA Molecular-Scale Detector against in Vivo Degradation for Intracellular miRNA Detection.

We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedra (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular target miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing a controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedra with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. The Tetra-ES detector exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the nonamplification molecular beacon counterpart and was capable of discriminating the miRNA target from the corresponding family members. The expression levels of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis, and therapeutics.