UV Cross-linking Research Articles

A facile method for cross-linking of methacrylated wood fibers for engineered wood composites

Chemical modifications are widely used to enhance the properties of wood composites and create a strong bonding mechanism for enhancing the dimensional stability, water resistance as well as decreasing carcinogenic formaldehyde emission. Esterification is the most-known modification way to enhance the durability of wood composites, but it does not improve mechanical performance. In this work, we demonstrated a two-step, easy and quick wood surface modification strategy based on microwave heating and UV crosslinking. Firstly, the fiber surface was reacted with methacrylic anhydride, then using methacrylated groups on wood, the fibers are covalently linked. As a proof-of-concept the fibers cross-linked within five minutes under UV radiation using benzophenone solution. Then, the effect of crosslinked wood fiber on the properties of mechanical and swelling of fiberboard were studied. Using SEM, FTIR-ATR, and swelling tests, we investigated the wood-based products' reaction mechanism, morphology, and internal bonding strength. The chemical cross-linking gives stronger bonding, compared to hydrogen bonding, between fibers even in wet conditions, resulting in a cross-linked foam-like structure. Also, wood panels were fabricated, compared to unmodified fibers, the internal bond strength and dimensional stability of fiberboards increased slightly. Overall, these results show that chemical cross-linking of wood fibers can be a fast and promising way to produce multi-functional wood composites.

Impact of Hierarchical Cation-Exchange Membranes' Chemistry and Crosslinking Level on Electrodialysis Demineralization Performances of a Complex Food Solution.

Hierarchical cation-exchange membranes (hCEMs) fabricated by blade coating and UV crosslinking of ionomer on top of a porous substrate demonstrated promising results in performing NaCl demineralization. In the food industry, complex solutions are used and hCEMs were never investigated before for these food applications. The performances of two different coating chemistries (urethane acrylate based: UL, and acrylic acid based: EbS) and three crosslinking degrees (UL5, UL6, UL7 for UL formulations, and EbS-1, EbS-2, EbS-3 for EbS formulations) were formulated. The impacts of hCEMs properties and crosslinking density on whey demineralization performances by electrodialysis (ED) were evaluated and compared to CMX, a high performing CEM for whey demineralization by ED. The crosslinking density had an impact on the hCEMs area specific resistance, and on the ionic conductance for EbS membrane. However, 70% demineralization of 18% whey solution was reached for the first time for hCEMs without any fouling observed, and with comparable performances to the CMX benchmark. Although some properties were impacted by the crosslinking density, the global performances in ED (limiting current, demineralization duration, global system resistance, energy consumption, current efficiency) for EbS and UL6 membranes were similar to the CMX benchmark. These promising results suggest the possible application of these hCEMs (UL6, EbS-2, and EbS-3) for whey demineralization by ED and more generally complex products as an alternative in the food industry.

Interfacial Ultraviolet Cross-Linking of Green Bilayer Dielectrics.

Electronic waste is a growing challenge which needs to be addressed through the integration of high-performance sustainable materials. Green dielectric polymers such as poly(vinyl alcohol) (PVA) have favorable electrical properties but are challenging to integrate into thin film electronics due to their physical properties. For example, PVA suffers from poor film formation and is hygroscopic. Bilayer dielectrics with interfacial cross-linking can enable the use of high-performance PVA with favorable surface chemistry by using a hydrophobic poly(caprolactone) (PCL) layer. In this study, we developed a benzodioxinone-terminated PCL layer, which can be UV cross-linked to the hydroxy groups of the PVA dielectric. This air-stable UV-cross-linking PCL dielectric was able to effectively cross-link with PVA, leading to high-performance capacitors and single-walled carbon nanotube-based thin film transistors. This UV cross-linking PCL dielectric led to significant improvements in shelf-life, ease of processing, and similar device performance compared to our previously reported thermally cross-linking PCL layer. The UV cross-linking at the interface between these bilayers can allow for the integration of high-speed roll-to-roll processing, which enables low-cost, sustainable, and high-performance electronics.

Robust Construction of Supersmall Zwitterionic Micelles Based on Hyperbranched Polycarbonates Mediates High Tumor Accumulation

Despite the numerous advantages of nanomedicines, their therapeutic efficacy is hampered by biological barriers, including fast in vivo clearance, poor tumor accumulation, inefficient penetration, and cellular uptake. Herein, cross-linked supersmall micelles based on zwitterionic hyperbranched polycarbonates can overcome these challenges for efficiently targeted drug delivery. Biodegradable acryloyl/zwitterion-functionalized hyperbranched polycarbonates are synthesized by a one-pot sequential reaction of Michael-type addition and ring-opening polymerization, followed by controlled modification with carboxybetaine thiol. Cross-linked supersmall zwitterionic micelles (X-CBMs) are readily prepared by straightforward self-assembly and UV cross-linking. X-CBMs exhibit prolonged blood circulation because of their cross-linked structure and zwitterion decoration, which resist protein corona formation and facilitate escaping RES recognition. Combined with the advantage of supersmall size (7.0 nm), X-CBMs mediate high tumor accumulation and deep penetration, which significantly enhance the targeted antitumor outcome against the 4T1 tumor model by administration of the paclitaxel (PTX) formulation (X-CBM@PTX).

Lipoic acid-based vitrimer-like elastomer.

Dynamic covalent networks (DCNs) are materials that feature reversible bond formation and breaking, allowing for self-healing and recyclability. To speed up the bond exchange, significant amounts of catalyst are used, which creates safety concerns. To tackle this issue, we report the synthesis of a lipoic acid-based vitrimer-like elastomer (LAVE) by combining (i) ring-opening polymerization (ROP) of lactones, (ii) lipoic acid modification of polylactones, and (iii) UV crosslinking. The melting temperature (Tm) of LAVE is below room temperature, which ensures the elastic properties of LAVE at service temperature. By carefully altering the network, it is possible to tune the Tm, as well as the mechanical strength and stretchability of the material. An increase in polylactone chain length in LAVE was found to increase strain at break from 25% to 180% and stress at break from 0.34 to 1.41 MPa. The material showed excellent network stability under cyclic strain loading, with no apparent hysteresis. The introduction of disulfide bonds allows the material to self-heal under UV exposure, extending its shelf life. Overall, this work presents an environmentally friendly approach for producing a sustainable elastomer that has potential for use in applications such as intelligent robots, smart wearable technology, and human-machine interfaces.

Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries.

UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.

Streamlined Purification of RNA-Protein Complexes Using UV Cross-Linking and RNA Antisense Purification.

RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization between biotinylated DNA probes and a target RNA. Target protein:RNA:DNA complexes are enriched by capture on streptavidin magnetic beads and purified through several denaturing washes that remove nonspecific protein and nucleic acid interactors. Mass spectrometry is used to identify proteins that are specifically enriched in the target RNA capture. This method has been applied to discover the protein interactions of noncoding RNAs but can be used to capture any RNA where the target sequence is known.

Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

Studying miRNA-mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein.

Transcriptome-wide analysis of RNA-binding partners is commonly achieved using UV crosslinking and immunoprecipitation (CLIP). Individual-nucleotide-resolution CLIP (iCLIP)enables identification of the specific position of the protein-RNA interaction. In addition to RNA-binding proteins (RBPs), microRNA (miRNA)-mRNA interactions also play a crucial role in the regulation of gene expression. Argonaute-2 (Ago2) mediates miRNA binding to a multitude of mRNA target sites, enabling the identification of miRNA-mRNA interactions by employing modified Ago2-CLIP protocols. Here, we describe an Ago2-specific CLIP protocol optimized for the use of small quantities of cell material, targeting endogenous Ago2 while avoiding possible methodological biases such as metabolic labeling or Ago2 overexpression and applying the latest advances in CLIP library preparation, the iCLIP2 protocol. In particular, we focus on the optimization of lysis conditions and improved radioactive labeling of the 5' end of the miRNA.

Injectable double-network hydrogel for corneal repair

Injuries to the cornea can cause corneal opacity or even blindness. Especially scald wounds and chemical burns to the cornea can usually lead to loss of corneal epithelium function and deep damage to the corneal stroma. Injectable hydrogel has been widely applied in corneal tissue engineering because it is easy to handle and can completely fill the defect area with minimally invasive surgical procedures. To reduce the risk and complications of penetrating keratoplasty and lamellar keratoplasty and to meet the demand for donated cornea, an injectable double-network (IDN) hydrogel based on methacrylated gelatin (Gel-MA) and oxidized hyaluronic acid (OHA) was constructed for focal corneal defect repairing. The Gel-MA/OHA IDN hydrogel had an interconnected porous microstructure and tunable mechanical properties with a modulus between 3.4 and 176.4 kPa. The hydrogel could promote cell adhesion and proliferation and afford the sustained release of hydrophilic/hydrophobic drugs. In-vivo corneal repair was performed by injecting the precursors into the pre-drilled epithelial-stromal defects of New Zealand White rabbits followed by immediate in-situ UV crosslinking. The results showed that the hydrogel had a strong capacity to promote corneal regeneration. Optical coherence tomography, slip lamp, fluorescein staining, scanning electron microscopy (SEM), and haematoxylin & eosin (H&E) staining were employed to illustrate the effectiveness and safety of corneal tissue regeneration. All the results showed that the optimized Gel-MA/OHA IDN hydrogel possessed excellent biocompatibility and could promote cornea repair in vivo. This study suggests that the injectable biocompatible double network hydrogel may provide a good choice for corneal repair and regeneration.

Alternative splicing of HSPA12A pre-RNA by SRSF11 contributes to metastasis potential of colorectal cancer.

Dysregulation of alternative splicing (AS) induced by serine/arginine-rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine-rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown. The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA-seq technology was used to analyze and screen SRSF11-triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini-gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co-IP) and Phospho-tag SDS-PAGE experiments were used to investigate PAK5-mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction. In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro-metastatic role in vitro and in vivo. By screening SRSF11-regulated AS events, we identified the binding motif of SRSF11-triggered splicing-switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N-cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation. SRSF11 exerts pro-metastatic effects in CRC by inhibiting the AS of HSPA12A pre-RNA. Our findings point to SRSF11-regulated HSPA12A splicing as a novel relationship between SRSF11-regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC.

Pd Nanoparticle-Loaded Smart Microgel-Based Membranes as Reusable Catalysts.

In this work, palladium-loaded smart membranes made by UV cross-linking of thermoresponsive microgels are prepared to obtain a reusable, catalytically active material which can, for example, be implemented in chemical reactors. The membranes are examined with respect to their coverage of a supporting mesh via atomic force microscopy measurements. Force indentation mapping was performed in the dried, collapsed state and in the swollen state in water to determine the Young modulus. Furthermore, we compare the catalytic activity of the membrane with the corresponding suspended colloidal nanoparticle microgel hybrids. For this purpose, the reduction of 4-nitrophenol is an established model reaction to quantify the catalytic activity by UV-vis spectroscopy. The membrane is embedded inside a continuous stirred tank reactor equipped for continuous monitoring of the reaction progress. Although catalysis with membranes shows lower catalytic activity than freely dispersed particles, membranes allow straightforward separation and recycling of the catalyst. The fabricated membranes in this work show no decrease in catalytic activity between several cycles, unlike free particles. The feasible and durable deposition of catalytically active inter-cross-linked microgel particles on commercial nylon meshes as supporting scaffolds, as demonstrated in this work, is promising for up-scaling of continuous industrial processes.

Genetically encoded chemical crosslinking of RNA in vivo.

Protein-RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein-RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

Особенности ультраструктурных изменений роговичного коллагена экспериментальных животных после уф кросслинкинга роговицы

Purpose. Electron microscopy analysis of collagen fibrils in the corneal stroma of rats after UV cross-linking. Material and methods. Experiments were carried out on rats in 2 groups: 1st – control (intact), 2nd – experiment (UV corneal cross-linking with riboflavin). The observation period is 3, 90 days. The UV irradiation power of the cornea is 3 mW / cm², the duration is 10 minutes. For electron microscopic examination, corneal pieces were fixed in a solution of 2 % glutaraldehyde. Dehydration and pouring were carried out according to the generally accepted method. Ultrathin sections were obtained using the ultramicrotome "Leica EM UC7" (Leica Microsystems) and viewed in the electron microscope JEM-1011 (Jeol). To determine the density of fibrillar packing of corneal collagen fibers, microphotographs were analyzed at magnification ×15,000. The number of collagen fibrils on an area of 250,000 nm² was calculated. The same microphotographs were used to measure the diameter of collagen fibrils. All numerical data were processed by the method of variational statistics. Histograms of the normal diameter distribution of collagen fibrils were analyzed. Results. The ultrastructure of corneal collagen on the 3rd day after UV corneal crosslinking was characterized by an increase in the interfibrillary space. This indicator was partially restored to its original values on the 90th day. At the same time, the fibrils retained the correct shape and sequence. The results of the analysis of the normal distribution of the diameter of collagen fibrils indicated an increase of an average of 10 %. Conclusion. An increase in the interfibrillary space after UV corneal crosslinking is probably associated with the development of postoperative stroma edema, which can be observed up to several months at the ultrastructural level. The revealed increase in the diameter of collagen fibrils is one of the factors increasing the strength and mechanical properties of the cornea. Keywords: UV cross-linking of rat cornea, electron microscopy, diameter of collagen fibrils, interfibrillary space.

In situ measurement of the stiffness increase in the posterior sclera after UV-riboflavin crosslinking by optical coherence elastography.

Scleral crosslinking may provide a way to prevent or treat myopia by stiffening scleral tissues. The ability to measure the stiffness of scleral tissues in situ pre and post scleral crosslinking would be useful but has not been established. Here, we tested the feasibility of optical coherence elastography (OCE) to measure shear modulus of scleral tissues and evaluate the impact of crosslinking on different posterior scleral regions using ex vivo porcine eyes as a model. From measured elastic wave speeds at 6 - 16 kHz, we obtained out-of-plane shear modulus value of 0.71 ± 0.12 MPa (n = 20) for normal porcine scleral tissues. After riboflavin-assisted UV crosslinking, the shear modulus increased to 1.50 ± 0.39 MPa (n = 20). This 2-fold change was consistent with the increase of static Young's modulus from 5.5 ± 1.1 MPa to 9.3 ± 1.9 MPa after crosslinking, which we measured using conventional uniaxial extensometry on tissue stripes. OCE revealed regional stiffness differences across the temporal, nasal, and deeper posterior sclera. Our results show the potential of OCE as a noninvasive tool to evaluate the effect of scleral crosslinking.

Injectable Hybrid-Crosslinked Hydrogels as Fatigue-Resistant and Shape-Stable Skin Depots.

Injectable hydrogels have gained considerable attention, but they are typically mechanically weak and subject to repeated physiological stresses in the body. Herein, we prepared polyurethane diacrylate (EPC-DA) hydrogels, which are injectable and can be photocrosslinked into fatigue-resistant implants. The mechanical properties can be tuned by changing photocrosslinking conditions, and the hybrid-crosslinked EPC-DA hydrogels exhibited high stability and sustained release properties. In contrast to common injectable hydrogels, EPC-DA hydrogels exhibited excellent antifatigue properties with >90% recovery during cyclic compression tests and showed shape stability after application of force and immersion in an aqueous buffer for 35 days. The EPC-DA hydrogel formed a shape-stable hydrogel depot in an ex vivo porcine skin model, with establishment of a temporary soft gel before in situ fixing by UV crosslinking. Hybrid crosslinking using injectable polymeric micelles or nanoparticles may be a general strategy for producing hydrogel implants resistant to physiological stresses.

Ceramic Omnidirectional Bioprinting in Cell-laden Suspensions for the Generation of Bone Analogs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

Tic12, a 12-kDa essential component of the translocon at the inner envelope membrane of chloroplasts in Arabidopsis.

The complexes translocon at the outer envelope membrane of chloroplasts and translocon at the inner envelope membrane of chloroplasts (TIC) mediate preprotein translocation across the chloroplast outer and inner envelope membranes, respectively. Tic20, Tic56, Tic100, and Tic214 form a stable one-megadalton TIC whose function is essential for Arabidopsis thaliana and Chlamydomonas reinhardtii. Tic20 plays a central role in preprotein translocation by forming a protein-conducting channel. Tic56, Tic100, and Tic214 are also indispensable for TIC function, but whether other components are required for this process remain unclear. Here, we demonstrate that a 12-kDa protein named Tic12 is part of the TIC in A. thaliana and participates in preprotein translocation across the inner envelope membrane. Tic12 was tightly associated with the TIC but disassociated under high-salt conditions in combination with Triton X-100. Site-specific UV crosslinking experiments revealed that Tic12 and Tic20 directly interact with the transit peptide of a translocating preprotein. The tic12 null mutants are albino and seedling lethal, similar to the other tic null mutants. Tic12 and Tic20 were also involved in preprotein translocation in (Pisum sativum) pea chloroplasts. Thus, Tic12 is an essential constituent that forms the functional core together with Tic20 in the one-megadalton TIC.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.