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[Methods in Molecular Biology] Plant Long Non-Coding RNAs Volume 1933 (Methods and Protocols) || Trimolecular Fluorescence Complementation (TriFC) Assay for Visualization of RNA-Protein Interaction in Plants
摘要： RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.
关键词： RNA-protein interaction,Long noncoding RNA,TriFC,Tobacco transient expression,In vivo visualization
更新于2025-11-21 11:08:12
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Dual-Signal Amplification Strategy for miRNA Sensing with High Sensitivity and Selectivity by Use of Single Au Nanowire Electrodes
摘要： MicroRNAs (miRNAs) have been applied as biomarkers and better detection of their expression profiles plays important roles in early diagnosis of cancers. In this work, a simple dual-signal amplification strategy has been used to construct a novel nanosensor on single Au nanowire electrodes (SAuNWEs) for miRNA-16 detection based on the “signal-on” and “signal-off” features during hybridization/de-hybridization process. The ferrocene-labeled aptamer capture probe (Fc-CP-16) is designed to hybridize with thiolated methylene blue-labeled DNA probe (MB-CP) on SAuNWE to form duplex DNA, and the addition of miRNA-16 can lead to the dissociation of duplex structure due to the highly matched sequences between miRNA-16 and Fc-CP-16. The remaining MB-CP can thus tend to recover its hairpin structure at the presence of Mg2+ through the hybridization of its complementary sequences. During this hybridization/de-hybridization process, the changes of Fc and MB oxidation peaks can be recorded, and there has a linear relationship between the sum of dual-signal changes (ΔI = ΔIMB + |ΔIFc|) and the logarithm of miRNA-16 concentrations, which can be used to detect miRNA-16. Including miRNA extraction, the dual-signal amplification strategy for miRNA sensing assay was carried out about 2 hours for the detection in real samples. This novel nanosensor has small dimension, good selectivity, rapid response and regeneration ability, which can satisfy the need for early cancer marker detection in cells/organelles.
关键词： nanosensors,single Au nanowire electrodes,dual-Signal Amplification Strategy,micro RNA
更新于2025-11-14 17:03:37
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Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH) to detect RNA in tissue: Simple and fast tissue RNA diagnostics
摘要： FISH-based RNA detection in paraffin-embedded tissue can be challenging, with complicated procedures producing uncertain results and poor image quality. Here, we developed a robust RNA detection method based on graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) in formalin-fixed paraffin-embedded (FFPE) tissues. Using a fluorophore-labeled peptide nucleic acid (PNA) attached to GO, the endogenous long noncoding RNA BC1, the constitutive protein β-actin mRNA, and miR-124a and miR-21 could be detected in the cytoplasm of a normal mouse brain, primary cultured hippocampal neurons, an Alzheimer’s disease model mouse brain, and glioblastoma multiforme tumor tissues, respectively. Coding and non-coding RNAs, either long or short, could be detected in deparaffinized FFPE or frozen tissues, as well as in clear lipid-exchanged anatomically rigid imaging/immunostaining-compatible tissue hydrogel (CLARITY)-transparent brain tissues. The fluorescence recovered by G-FISH correlated highly with the amount of miR-21, as measured by quantitative real time RT-PCR. We propose G-FISH as a simple, fast, inexpensive, and sensitive method for RNA detection, with a very low background, which could be applied to a variety of research or diagnostic purposes.
关键词： glioblastoma multiforme tumor,tissue RNA diagnostics,Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH),Alzheimer’s disease,formalin-fixed paraffin-embedded (FFPE) tissue
更新于2025-09-23 15:23:52
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Light-Inducible Exosome-Based Vehicle for Endogenous RNA Loading and Delivery to Leukemia Cells
摘要： Exosomes are a novel and promising drug delivery platform because of their endogenous origin, stability, biocompatibility, and other unique features. As the efficient loading and delivery of long RNA to target cells for therapeutic purposes remains challenging, a new exosome-based RNA delivery system is proposed using a controllable RNA enrichment and releasing protocol. The system employs RNA aptamer–protein interactions and reversible light-inducible protein–protein interaction modules by remolding exosome producer cells. Endogenous microRNA 21 (miR-21) sponges, inhibitors of miR-21, are successfully enriched on the plasma membrane and are sorted into exosomes by the biogenesis of the exosomes. The loading capacity of miR-21 sponges is enhanced by 14-fold in the light-inducible loading system. In addition, targeted delivery of miR-21 to leukemia cells is achieved by modifying exosomes with the cholesterol-conjugated aptamer AS1411, resulting in significant cell apoptosis by blocking the function of miR-21 in leukemia cells. This work provides an exosome-based light-inducible vehicle to efficiently load and deliver long endogenous RNA, which can enable more RNA-based therapeutics for personalized cancer medicine.
关键词： miRNA sponge,leukemia,RNA enrichment,exosomes,reversible light-inducible vehicles,aptamers
更新于2025-09-23 15:23:52
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Ruthenium(II) polypyridyl complex [Ru(phen)2dppz-idzo]2+ as a colorimetric molecular “light switch” and powerful stabilizer for the RNA triplex poly(U)·poly(A)*poly(U)
摘要： The interaction of [Ru(phen)2dppz-idzo]2+ (phen = 1,10-phenanthroline, dppz-idzo = dppz-imidazolone) with triplex RNA poly(U)?poly(A)*poly(U) was carried out by using spectroscopic and viscometric techniques in this work. Luminescent titrations suggest that [Ru(phen)2dppz-idzo]2+ shows better selectivity for poly(U)?poly(A)*poly(U) compared with poly(U)?poly(A) and poly(U), this complex exhibits a “light switch” effect with an emission enhancement factor of about 123 in the presence of poly(U)?poly(A)*poly(U). Significantly, this “light switch” behavior could even be observed by the naked eye under irradiation with UV light. To our knowledge, [Ru(bpy)2dppz-idzo]2+ is the first small molecule able to serve as a colorimetric molecular “light switch” for the triplex poly(U)?poly(A)*poly(U). Combined with the spectral and viscometric results as well as [Ru(phen)2dppz-idzo]2+ stabilizing the template duplex poly(U)?poly(A), we speculate that [Ru(phen)2dppz-idzo]2+ prefers to bind with the Hoogsteen base-paired strand (the third strand) of the triplex, thus the intercalating [Ru(phen)2dppz-idzo]2+ stabilizing the third strand is more marked in comparison with the Watson-Crick base-paired duplex of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)?poly(A)*poly(U) with small molecule, particularly ruthenium(II) complexes.
关键词： Ru(II) complexes,Stabilization,Triplex RNA,Colorimetic molecular light switch
更新于2025-09-23 15:23:52
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HMGB1 siRNA can reduce damage to retinal cells induced by high glucose in vitro and in vivo
摘要： Background: Diabetic retinopathy (DR), one of the most common complications of late-phase diabetes, is associated with many risk factors, among which continuous low-grade inflammation is one of the principal ones. As such, lowering inflammation levels and maintain the viability of human retinal endothelial cells (HRECs) are critical for DR therapy. HMGB1 is a well-known proinflammatory cytokine. However, whether HMGB1 small interfering RNA (siRNA) can protect retina cells under a high-glucose environment from morphological changes and functional abnormalities remain undetermined. We aimed to investigate the effect of HMGB1 siRNA on retinal cells in DR. Materials and methods: A total of 80 adult Wistar rats were randomly divided into four groups (n=20 each): normal control, diabetes mellitus (DM), scrambled (Scr) siRNA, and HMGB1 siRNA. Rats in the DM, Scr siRNA, and siRNA groups were established by intraperitoneal injection of streptozotocin. At 16 weeks after injection, rats in the siRNA and Scr-siRNA groups were intravitreally injected with 2 μL HMGB1 siRNA and 2 μL Scr-siRNA, while rats in the control and DM groups were intravitreally injected with the same dose of sterile saline. At 1 week after injections, we performed the following experiments. Immunohistochemical staining and real-time quantitative polymerase chain reaction were performed to test HMGB1 protein and messenger RNA expression in retinas. We performed TUNEL assays to detect retinal cell apoptosis and electroretinography to detect retinal function. In HRECs treated with high glucose, proliferation, morphology, apoptosis, superoxide dismutase (SOD), and reactive oxygen species production were detected. Western blot was applied to determine the expressions of HMGB1 and its related protein and apoptosis protein. Results: Intravitreal injection of HMGB1 siRNA reduced protein and messenger RNA expression of HMGB1 (both P,0.05). Intravitreal injection of HMGB1 siRNA reduced apoptosis of retinal cells (P,0.05), protected morphological changes in the retina, and improved the function of the retina (P,0.05). In HRECs treated with high glucose, HMGB1 siRNA pretreatment increased cell viability, reduced cell apoptosis, and reduced oxidative damage to cells (all P,0.05). Western blot detection found that HMGB1 siRNA pretreatment can inhibit the expression of cleaved caspase 3 and improve the expression of BCL2 (P,0.05). HMGB1 and NFκB expression increased in a time-dependent manner in the high-glucose environment and IKKβ and NFκB protein expression decreased significantly after HMGB1 silencing. Conclusion: As a therapeutic target, HMGB1 siRNA can reduce retinal cell damage induced by high glucose in vitro and in vivo and delay DR progress through the HMGB1–IKKβ–NFκB signaling pathway.
关键词： small interfering RNA,diabetic retinopathy,human retinal endothelial cells,inhibitor of nuclear factor κB,nuclear factor κB,high-mobility group box 1
更新于2025-09-23 15:22:29
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Dopamine Binding and Analysis in Undiluted Human Serum and Blood by the RNA-Aptamer Electrode
摘要： Specific analysis of such neurotransmitters as dopamine by the aptamer electrodes in biological fluids is detrimentally affected by non-specific adsorption of media, particularly pronounced at positive charges of the electrode surface at which dopamine oxidizes. Here, we show that dopamine analysis at the RNA-aptamer/cysteamine-modified electrodes is strongly inhibited in undiluted human serum and blood due to non-specific interfacial adsorption of serum and blood components. We demonstrate that non-specific adsorption of serum proteins (but not of blood components) could be minimized when analysis is performed in a flow and injections of serum samples are followed by washing steps in a phosphate buffer solution (PBS) carrier. Under those conditions, the dopamine-aptamer binding affinity in whole human serum of (1.9±0.3)×104 M-1 s-1 was comparable to (3.7±0.3)×104 M-1 s-1 found in PBS, and the dopamine oxidation signal linearly depended on the dopamine concentration, providing the sensitivity of analysis of 73 ± 3 nA μM-1 cm-2 and the LOD of 114 ± 8 nM. The flow-injection apatmer-electrode system was used for direct analysis of basal levels of dopamine in undiluted human serum samples, without using any physical separators (membranes) or filtration procedures. The results suggest a simple strategy for combatting biosurface fouling most pronounced at positive electrode potentials and assist in designing more efficient antifouling strategies for biomedical applications.
关键词： Human serum,Blood,Surface fouling,RNA aptamer electrode,Dopamine,Chronoamperometry,Electrochemical Impedance,Flow-through cell
更新于2025-09-23 15:22:29
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[Institution of Engineering and Technology Fifth Asia International Symposium on Mechatronics (AISM 2015) - Guilin, China (7-10 Oct. 2015)] Fifth Asia International Symposium on Mechatronics (AISM 2015) - Enhanced plasmonic effects in Ag decorated amorphous TiO2 nanotube arrays
摘要： The process of converting raw RNA sequencing (RNA-seq) data to interpretable results can be circuitous and time-consuming, requiring multiple steps. We present an RNA-seq mapping algorithm that streamlines this process. Our algorithm utilizes a hash table approach to leverage the availability and the power of high memory machines. SNAPR, which can be run on a single library or thousands of libraries, can take compressed or uncompressed FASTQ and BAM ?les, and output a sorted BAM ?le, individual read counts, and gene fusions, and can identify exogenous RNA species in a single step. SNAPR also does native Phred score ?ltering of reads. SNAPR is also well suited for future sequencing platforms that generate longer reads. We show how we can analyze data from hundreds of TCGA samples in a matter of hours while identifying gene fusions and viral events at the same time. With the reference genome and transcriptome undergoing periodic updates and the need for uniform parameters when integrating multiple data sets, there is great need for a streamlined process for RNA-seq analysis. We demonstrate how SNAPR does this ef?ciently and accurately.
关键词： computational biology,biology,biology computing,RNA.,genetic expression,Bioinformatics
更新于2025-09-23 15:21:01
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A UV cross-linking method combined with infrared imaging to analyse RNA–protein interactions
摘要： Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [c-32P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modi?ed UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDyeVR 800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the OdysseyVR Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the ef?ciency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facili- tating quantitative comparisons. To validate the method, we have con?rmed the binding of HuD to the 30-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer’s disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant puri?ed glutathione-S-transferase–HuD fu- sion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease.
关键词： Tau,RNA-binding protein,UV cross-linking,neurodegeneration,HuD,OdysseyVR
更新于2025-09-23 15:19:57
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RNA-Seq analysis revealed the molecular mechanisms of photobiomodulation effect on human fibroblasts
摘要： Background The Photobiomodulation (PBM) effect has been applied to various clinical therapy for a long time. However, the mechanism related to the PBM effect in terms of wavelengths has been lack of in-depth study, except that ultraviolet radiation has attracted much attention due to its strong cell killing effect. Purpose To clarify the principle behind PBM and the main mechanism of improvement. Methods To carry on this study, we created light equipment using three LED chips, which emit 390 nm ultraviolet radiation, 415 nm blue light and 660 nm red light, respectively. We choose human fibroblasts (HF) to be irradiated by three different wavelengths for PBM test. In this study, we used cell counting kit (CCK-8) test to show the cell proliferation roughly and reported on a systematic RNA sequencing (RNA-seq) analysis at transcriptional expression levels from HF, which accepted PBM of different wavelengths of light. Results We found that 415 nm blue light inhibited cell proliferation and 660 nm red light stimulated cell proliferation while 390 nm ultraviolet radiation has little influence on cell proliferation. Furthermore, RNA-seq results showed that CSF1R, PPP3CC, ITGAL, ITGAM, IL2RB and several other differentially expressed genes (DEGs) are involved in the cell proliferation. Relative DEGs values for matrix metalloproteinases (MMPs) gene family has shown a great difference in blue and red light radiation especially on MMP25, MMP9, MMP21 and MMP13. Conclusion Taken together, the results provide a valuable resource to describe the variation of HFs under PBM of different light at gene level.
关键词： MMPs,RNA-Seq,Human fibroblasts(HF),Photobiomodulation (PBM),LED
更新于2025-09-23 15:19:57