DNA-dependent RNA polymerases in plants.

DNA-dependent RNA polymerases (Pols) transfer the genetic information stored in genomic DNA to RNA in all organisms. In eukaryotes, the typical products of nuclear Pol I, Pol II, and Pol III are ribosomal RNAs, mRNAs, and transfer RNAs, respectively. Intriguingly, plants possess two additional Pols, Pol IV and Pol V, which produce small RNAs and long noncoding RNAs, respectively, mainly for silencing transposable elements. The five plant Pols share some subunits, but their distinct functions stem from unique subunits that interact with specific regulatory factors in their transcription cycles. Here, we summarize recent advances in our understanding of plant nucleus-localized Pols, including their evolution, function, structures, and transcription cycles.

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Rice requires a chromatin remodeler for Polymerase IV-small interfering RNA production and genomic immunity.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

Functionalized DNA Origami-Enabled Detection of Biomarkers.

Biomarkers are crucial physiological and pathological indicators in the host. Over the years, numerous detection methods have been developed for biomarkers, given their significant potential in various biological and biomedical applications. Among these, the detection system based on functionalized DNA origami has emerged as a promising approach due to its precise control over sensing modules, enabling sensitive, specific, and programmable biomarker detection. We summarize the advancements in biomarker detection using functionalized DNA origami, focusing on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and applications in disease diagnosis and monitoring. These applications are organized into sections based on the type of biomarkers - nucleic acids, proteins, small molecules, and ions - and concludes with a discussion on the advantages and challenges associated with using functionalized DNA origami systems for biomarker detection.

The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice.

RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of RNA polymerase V (Pol V), a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long noncoding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V-independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V-dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V-dependent loci but not on Pol V-independent loci. Our results reveal the function of rice Pol V for long noncoding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids.

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

miRNA Detection for Prostate Cancer Diagnosis by miRoll-Cas: miRNA Rolling Circle Transcription for CRISPR-Cas Assay.

Micro-RNA (miRNA) emerges as a promising type of biomarker for cancer diagnosis. There is an urgent need for developing rapid, convenient, and precise miRNA detection methods that may be conducted with limited laboratory facilities, especially in underdeveloped areas. Herein, we developed a miRNA detection method termed miRoll-Cas, where miRNA is first amplified by rolling circle transcription and then subject to CRISPR-Cas13a assay. Using miRoll-Cas, we realized the sensitive detection of multiple cancer-relevant miRNA markers (miR21, miR141, and Let7b) and specifically identified other variants of the Let7 family, which can accurately discriminate prostate cancer patients from healthy people. We envision that miRoll-Cas may be readily translated to clinical applications in the diagnosis of a variety of diseases beyond cancer.

Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement.

Detection of viral infections (e.g., SARS-CoV-2) with high precision is critical to disease control and treatment. There is an urgent need to develop point-of-care detection methods to complement the gold standard laboratory-based PCR assay with comparable sensitivity and specificity. Herein, we developed a method termed mCAD to achieve ultraspecific point-of-care detection of SARS-CoV-2 RNA while maintaining high sensitivity by programming multiplex rolling circle amplification and toehold-mediated strand displacement reactions. RCA offers sufficient amplification of RNA targets for subsequent detection. Most importantly, a multilayer of detection specificity is implemented into mCAD via sequence-specific hybridization of nucleic acids across serial steps of this protocol to fully eliminate potential false-positive detections. Using mCAD, we demonstrated a highly specific, sensitive, and convenient visual detection of SARS-CoV-2 RNA from both synthetic and clinical samples, exhibiting performance comparable to qPCR. We envision that mCAD will find its broad applications in clinical prospects for nucleic acid detections readily beyond SARS-CoV-2 RNA.

Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice.

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well studied in Arabidopsis (Arabidopsis thaliana), most mutations in RdDM genes cause no remarkable developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 (OsRDR2) in rice (Oryza sativa). Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH (H = A, C, or T) methylation. Moreover, male and female reproductive development was disturbed, which led to sterility in osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation may regulate the expression of multiple key genes involved in stamen development, meiosis, and pollen viability. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of CHH methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity, which includes OsRDR2 activity. Our results, which revealed a global increase in CHH methylation through enhancement of RdDM activity in reproductive organs, suggest a crucial role for OsRDR2 in the sexual reproduction of rice.

Promoting Cell Fusion by Polyvalent DNA Ligands.

Artificially induced in vitro cell fusion is one essential technique that has been extensively used for biological studies. Nevertheless, there is a lack of robust and efficient method to produce fused cells efficiently. Herein, we proposed to use cell-membrane-anchored polyvalent DNA ligands (PDL) to bring cells into close proximity by forming clusters to enhance PEG-induced cell fusion. PDL of complementary sequences are separately anchored onto different population of cells through cholesterol-induced hydrophobic insertion into lipid membrane. Cells are clustered via mixing cells of complementary PDL prior to cell fusion. PDL exhibited strong stability on cell membrane, induced efficient cell clustering, and eventually achieved cell fusion efficiently in combination with PEG induction. We demonstrated homogeneous and heterogeneous cell fusion of high yield on various cell types. This report presented a programmable yet robust technique for achieving efficient cell fusion that hold great application potentials.

In Situ DNA Self-Assembly on the Cell Surface Drives Unidirectional Clustering of Membrane Proteins for the Modulation of Cell Behaviors.

Cell membrane proteins play a pivotal role in regulating intracellular signal transductions and cell behaviors. Many membrane proteins form clusters in order to initiate downstream signaling pathways for the modulation of cell behaviors. Developing rational methods to program the in situ clustering of designated membrane proteins on the cell surface to form large assemblies remains challenging. Here we use the membrane-anchored DNA hybridization chain reaction (HCR) to induce DNA self-assembly on the live cell surface and drive the unidirectional clustering of membrane proteins for the modulation of cell behaviors. Reactive DNA strands are specifically anchored onto the membrane proteins of interest by using DNA aptamers. Upon activation, the chain reaction between the protein-anchored DNA strands drives the assembly of membrane proteins forming one-dimensional clusters. We demonstrate both homogeneous and heterogeneous clustering of membrane proteins on multiple cell types that exhibit a potent capability for modulating cell behaviors including migration, proliferation, and survival.

Programming DNA Aptamer Arrays of Prescribed Spatial Features with Enhanced Bioavailability and Cell Growth Modulation.

Epithelial cell adhesion molecules (EpCAMs) play pivotal roles in tumorigenesis in many cancer types, which is reported to reside within nano- to microscale membrane domains, forming small clusters. We propose that building multivalent ligands that spatially patch to EpCAM clusters may largely enhance their targeting capability. Herein, we assembled EpCAM aptamers into nanoscale arrays of prescribed valency and spatial arrangements by using a rectangular DNA pegboard. Our results revealed that EpCAM aptamer arrays exhibited significantly higher binding avidity to MCF-7 cells than free monovalent aptamers, which was affected by both valency and spatial arrangement of aptamers. Furthermore, EpCAM aptamer arrays showed improved tolerance against competing targets in an extracellular environment and potent bioavailability and targeting specificity in a xenograft tumor model in mice. This work may shed light on rationally designing multivalent ligand complexes of defined parameters with optimized binding avidity and targeting capability toward various applications in the biomedical fields.

Spatially Reprogramed Receptor Organization to Switch Cell Behavior Using a DNA Origami-Templated Aptamer Nanoarray.

Receptor oligomerization is a highly complex molecular process that modulates divergent cell signaling. However, there is a lack of molecular tools for systematically interrogating how receptor oligomerization governs the signaling response. Here, we developed a DNA origami-templated aptamer nanoarray (DOTA) that enables precise programming of the oligomerization of receptor tyrosine kinases (RTK) with defined valency, distribution, and stoichiometry at the ligand-receptor interface. The DOTA allows for advanced receptor manipulations by arraying either monomeric aptamer ligands (mALs) that oligamerize receptor monomers to elicit artificial signaling or dimeric aptamer ligands (dALs) that preorganize the receptor dimer to recapitulate natural activation. We demonstrated that the multivalency and nanoscale spacing of receptor oligomerization coordinately influence the activation level of receptor tyrosine kinase signaling. Furthermore, we illustrated that DOTA-modulated receptor oligomerization could function as a signaling switch to promote the transition from epithelia to mesenchymal-like cells, demonstrating robust control over cellular behaviors. Together, we present a versatile all-in-one DNA nanoplatform for the systematical investigation and regulation of receptor-mediated cellular response.

First morning voided urinary gonadotropins in children: verification of method performance and establishment of reference intervals.

OBJECTIVES: Urinary luteinizing hormone (uLH) and urinary follicle-stimulating hormone (uFSH) have been shown to be useful screening and management tools for children with central precocious puberty. However, studies on uLH and uFSH reference intervals are scarce. Therefore, we aimed to establish reference intervals for uLH and uFSH, according to age, sex, and pubertal status in apparently healthy children aged 6-11 years. METHODS: We performed detection capability, precision, accuracy by recovery, linearity, agreement analysis, and stability testing to analyze the method performance of uLH and uFSH. The Clinical Laboratory Standards Institute's C28-A3 criteria was used to establish the reference intervals. RESULTS: Both uLH and uFSH were stable at 4 °C for 52.6 h and 64.8 days, respectively. The total imprecision of uFSH is within the manufacturer's claim, while the total imprecision of uLH remained within tolerable bias. Both uLH and uFSH could be measured with acceptable detection capability. The recovery rates of the hormones were 87.6-98.8% and 102.8-103.4%, respectively, and therefore within acceptable limits. There were significant correlations between the serum and urine concentrations (LH: r=0.91, p<0.001; FSH: r=0.90, p<0.001). The reference intervals of uLH and uFSH were established according to age, sex, and pubertal status. CONCLUSIONS: We established reference intervals for uLH and uFSH based on age, sex and pubertal status to provide a non-invasive clinical screening tool for precocious puberty in children aged 6-11 years.

Profiling and Regulating Proteins That Adsorb to DNA Materials in Human Serum.

DNA aptamers and framework DNA nanostructures are emerging DNA materials with many appealing biological applications including biosensing, bioimaging, drug delivery, and so forth. When placed in physiological fluids, they inevitably encounter biomolecules (majorly proteins) and form complexes that largely affect their biological fate. Nevertheless, little is known regarding the quantitative profile of proteins that adsorb to DNA aptamers and DNA nanostructures in biological environments, and there are no potent strategies to regulate protein profiles. Herein, we performed a proteomic analysis to profile proteins that bind to DNA aptamers (Sgc8c and SYLC3) and nanostructures (a tetrahedral DNA nanostructure and a DNA origami rod) in human serum using liquid chromatography-mass spectrometry (LC-MS). Dozens to hundreds of proteins were identified with each DNA material exhibiting highly distinctive profiles. It was also revealed that the origin of serum (from healthy donor vs from prostate cancer patients) causes significant differences in profiles of bound proteins. Furthermore, we demonstrated that the protein profile may be regulated by tethering a layer of single-stranded DNA (polythymine) onto the DNA origami rod to alleviate the adsorption of complement-associated proteins, which significantly reduced its sequestration by macrophages. Taken together, this study has provided qualitative and quantitative proteomic profiles regarding serum proteins that adsorb to various DNA materials and have demonstrated that the composition of interacted proteins may be regulated toward better biological performances.

Lower testosterone levels predict increasing severity and worse outcomes of hepatitis B virus-related acute-on-chronic liver failure in males.

BACKGROUND: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a serious liver disease with pathogenesis remaining unclear. This study aims to investigate the association between testosterone levels, stage (early, middle, or late, categorized according to clinical manifestation), severity scores, and clinical outcomes of HBV-ACLF. METHODS: This single-center observational study involved 160 male patients with HBV-ACLF, 151 chronic hepatitis B patients without liver failure (CHB) and 106 healthy controls (HC). Morning blood samples were collected and androgen levels analyzed by chemi-bioluminescent immunoassay. Time to death or liver transplantation within 90 days comprised the primary composite outcome. RESULTS: Serum levels of total testosterone (TT), free testosterone index (FTI), dehydroepiandrosterone sulfate and cortisol were significantly lower among HBV-ACLF than CHB and HC, while androstenedione was higher. Low TT, sex hormone binding globulin and FTI were associated with increased stage (of HBV-ACLF, ascites, and hepatic encephalopathy) and severity scores (Model for End-stage Liver Disease and Chinese Group on the Study of Severe Hepatitis B-ACLF scores). Low TT (< 142.39 ng/dL) was a risk factor for both the composite outcome and for death alone within 90 days. Multivariate analysis revealed TT to be an independent predictor for the composite outcome (hazard ratio 2.57, 95% CI 1.09-6.02; P = 0.030). CONCLUSION: Low serum testosterone is common among male patients with HBV-ACLF and predictive of increased severity and worse outcome of the disease and may play an important role in the progression of HBV-ACLF.

DNA Origami Guided Self-Assembly of Plasmonic Polymers with Robust Long-Range Plasmonic Resonance.

Plasmonic polymers consisting of metallic nanoparticles (NPs) are able to squeeze light into the deep-subwavelength space and transfer along a highly confined nanoscale path in long range. DNA nanotechnology, particularly benefiting from the molecular programmability of DNA origami, has provided otherwise nearly impossible platforms for constructing plasmonic nanoparticle polymers with designer configurations and nanoscale gaps. Here, we design and assemble a DNA origami hashtag tile that is able to polymerize into one-dimensional chains with high rigidity. The DNA origami hashtag chains are used as frames to enable robust, versatile, and precise arrangement of metallic NPs into micrometer-long chiral and magnetic plasmonic polymers, which are capable of efficiently transporting plasmonic angular momentum and magnetic surface plasmonic polaritons at the deep-subwavelength scale. Our work provides a molecular platform for the fabrication of long, straight, and structurally complex nanoparticle polymers with emerging plasmonic properties that are appealing to a variety of fields.

Programming Surface-Enhanced Raman Scattering of DNA Origami-templated Metamolecules.

DNA origami holds an unprecedented capability on assembling metallic nanoparticles into designer plasmonic metamolecules of emerging properties, including surface-enhanced Raman scattering (SERS). SERS metamolecules were produced by positioning nanoparticles in close proximity to each other on a DNA origami template for Raman enhancement. In earlier reports, SERS metamolecules were generally assembled into clusters containing small number of nanoparticles (2, 3, or 4) and thus had limited programmability over SERS. Herein, we expanded the structural complexity of SERS metamolecules by increasing the number of nanoparticles and by arranging them into sophisticated configurations. DNA origami hexagon tile was used as the assembling template to fabricate clusters consisting of 6, 7, 12, 18, and 30+ metallic nanoparticles. Programmable SERS was realized via controlling the size, number, or spatial arrangement of nanoparticles. We believe this method offers a general platform for fabricating sophisticated nanodevices with programmable SERS that may be applied to a variety of fields including plasmonics, nanophotonics, and sensing.

DNA nanostructures constructed with multi-stranded motifs.

Earlier studies in DNA self-assembly have foretold the feasibility of building addressable nanostructures with multi-stranded motifs, which is fully validated in this study. In realizing this feasibility in DNA nanotechnology, a diversified set of motifs of modified domain lengths is extended from a classic type. The length of sticky ends can be adjusted to form different dihedral angles between the matching motifs, which corresponds to different connecting patterns. Moreover, the length of rigidity core can also be tuned to result in different dihedral angles between the component helices of a certain motif therefore different numbers of component helices. The extended set of motifs is used for self-assembly of complex one dimensional, two dimensional and three dimensional structures.