N-hydroxysuccinimide Ester Research Articles

The Usage of an AMCA-Modified Aptamer Facilitates the Rapid Detection of Vibrio vulnificus

Background: Vibrio vulnificus can cause serious infections in human beings associated with the consumption of raw oysters or cuts exposed to seawater. The traditional method for culturing V. vulnificus is time-consuming and has a high failure rate. Objectives: This study aims to detect V. vulnificus using an AMCA-modified specific DNA aptamer. Methods: Common pathogenic microorganisms present in the seawater of the Fujian Sea area were collected, cultured, and identified. The samples were found to contain mainly V. vulnificus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, V. alginolyticus, V. parahaemolyticus, and V. cholerae. AMCA was conjugated with 5’ ends of the aptamer using N-hydroxysuccinimide ester (NHS ester) to target V. vulnificus and produce a fluorescent signal upon binding. The aptamer was screened and optimized for rapid detection of V. vulnificus. We collected the 50 bacterial strains isolated from clinical secretion samples and used a fluorescence microscope to determine whether the sample contained V. vulnificus or not. We compared these results with those obtained from VITEK MS (considered the gold standard) to test the sensitivity and specificity of the AMCA-modified aptamer using IBM SPSS Statistics 22. Results: In this experiment, the sensitivity and specificity of the modified aptamer for detecting V. vulnificus were determined to be 100% [95% CI (0.39, 1)] and 93.4% [95% CI (0.81, 0.98)], respectively. The positive predictive value was 57% [95% CI (0.20, 0.88)], and the negative predictive value was 100% [95% CI (0.89, 1)]. These findings indicate that V. vulnificus specimens can be rapidly detected via fluorescence reaction within 30 minutes. Conclusions: Our results suggest that this modified DNA aptamer has the potential to be used for diagnosing V. vulnificus. Further research is needed to explore the application of aptamers in pathogen infections.

Modular Synthesis of Methyl-Substituted Novel Psoralen N-Hydroxysuccinimide Esters and Evaluation of DNA Photocrosslinking Properties of the Corresponding Triplex-Forming Oligonucleotide Conjugates

AbstractPsoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been used to induce DNA mutations or to suppress gene expression through the formation of crosslinked products with DNA in a sequence-specific manner. Psoralen can crosslink with DNA at its furan-ring and/or pyrone-ring side, yielding either a monoadduct or diadduct (interstrand crosslinking) product. The differences in the crosslinked structures of Ps-TFOs with the target DNAs are closely related to the changes in the biological outcomes induced by the Ps-TFOs. However, only a few reports have discussed the photocrosslinking properties of Ps-TFOs. The photocrosslinking properties of Ps-TFOs with structurally diverse psoralen derivatives remain elusive. Herein, we report the modular synthesis of novel methyl-substituted psoralen N-hydroxysuccinimide (NHS) esters. By using these esters, the effect of the methyl substituent of psoralen on the photocrosslinking of the corresponding Ps-TFOs was examined. The amount of the diadduct product was significantly reduced in the presence of methyl substituents at the C-3 and C-4 positions, while the total amount of photocrosslinking product was maintained. This work demonstrates the possibility of controlling the crosslinked product of Ps-TFOs by introducing methyl groups into psoralen: this ability to manipulate the product is an important factor in the biological applications of Ps-TFOs.

Chemical acylation of pea protein isolate hydrolysate with fatty acid N-hydroxysuccinimide esters: Effect on structure and functional properties

Applications of pea protein in the food industry have been greatly restricted by its poor functional properties. In order to solve this problem, a novel technique combining enzymatic hydrolysis and fatty acid acylation has been applied in this work to construct a pea protein-fatty acid covalent complex that aims to improve its functional properties. The processed pea protein with increased water solubility tends to decrease the chance of self-aggregation. Additionally, emulsifying and antioxidant properties have also been found after this process. On top of that, the modified pea protein has been characterized by Fourier transform infrared and circular dichroism spectroscopy. These results demonstrate that these properties were mainly caused by the acylation of the amino group from hydrolyzed pea protein and the carboxyl group from the fatty acid. The enzymatic hydrolysis/fatty acid acylation research provides insights into manufacturing high-quality functional lipoproteins from inexpensive pea protein for the food industry.

Ultrasensitive electrochemical sensor for detection of salivary cortisol in stress conditions.

A natural stress response induces elevated cortisol levels in biological fluids, such as saliva. While current sensor technologies can detect cortisol in real time, their sensitivity and reliability for human subjects have not been assured. This is due to relatively low concentrations of salivary cortisol, which fluctuate throughout the day and vary significantly between individuals. To address these challenges, we present an improved electrochemical biosensor leveraging graphene's exceptional conductivity and physicochemical properties. A1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE-NHS)-modified commercial graphene foam (GF) electrode is presentedto realize an ultra-sensitive biosensor for cortisol detection directly in human saliva. The biosensor fabrication process entails the attachment of anti-cortisol monoclonal antibodies (mAb-cort) onto a PBASE-NHS/GF electrode through noncovalent immobilization on the vertically stratified graphene foam electrode surface. This unique immobilization strategy preserves graphene's structural integrity and electrical conductivity while facilitating antibody immobilization. The binding of cortisol to immobilized mAb-cort is read out via differential pulse voltammetry using ferri/ferro redox reactions. The immunosensor demonstrates an exceptional dynamic range of 1.0fgmL-1 to 10,000pgmL-1 (R2 = 0.9914) with a detection limit of 0.24fgmL-1 (n = 3) for cortisol. Furthermore, we have established the reliability of cortisol sensors in monitoring human saliva. We have also performed multiple modes of validation, one against the established enzyme-linked immunosorbent assay (ELISA) and a second by a third-party service Salimetric on 16 student volunteers exposed to different stress levels, showing excellent correlation (r = 0.9961). These findings suggest the potential for using mAb-cort/PBASE-NHS/GF-based cortisol electrodes for monitoring salivary cortisol in the general population.

Heterobifunctional Cross-Linker with Dinitroimidazole and N-Hydroxysuccinimide Ester Motifs for Protein Functionalization and Cysteine-Lysine Peptide Stapling.

A heterobifunctional cross-linker with one sulfhydryl-reactive dinitroimidazole end and another amine-reactive N-hydroxysuccinimide (NHS) ester end was designed and synthesized. The two motifs of this cross-linker, dinitroimidazole and NHS ester, proved to react with thiol and amine, respectively, in an orthogonal way. The cross-linker was further applied to construct stapled peptides of different sizes and mono- and dual functionalization (including biotinylation, PEGylation, and fluorescence labeling) of protein.

Label-free flexible immunosensor based on nitrogen-doped graphene with titanium dioxide for the determination of depression marker dendritic cell nuclear protein-1

In this work, a label-free flexible immunosensor for selective and sensitive detection of dendritic cell nuclear protein-1 (DCNP1), a newly discovered potential marker of depression, was successfully constructed. At first, a highly conductive nanomaterial nitrogen-doped graphene with titanium dioxide (NG/TiO2) was prepared using a one-pot method, and it was used as an electrode modification material in the construction of immunosensors for the first time. NG/TiO2 was functionalized with 1-pyrene-butyric acid N-hydroxysuccinimide ester (PSE), and then the DCNP1 antibody could be better immobilized on the surface of the modified indium tin oxide-polyethylene terephthalate (ITO-PET). Then bovine serum albumin (BSA) was used to block non-specific active sites, and the immunosensor (BSA/anti-DCNP1-PSE/NG/TiO2/ITO-PET) can be obtained. The fabricated NG/TiO2 and the construction process of the immunosensor were characterized by scanning electron microscopy, infrared spectroscopy, cyclic voltammetry, and electrochemical impedance methods. Differential pulse voltammetry (DPV) was used to measure different concentrations of DCNP1 after optimizing the ratio of NG and TiO2, the modification amount of NG/TiO2, the ratio of NG/TiO2 to PSE, the concentration of anti-DCNP1 and its immobilization time, the concentration of BSA, and the incubation time of DCNP1. The constructed sensor had a wide linear range of 1 ∼ 104 pg mL−1, and a low detection limit of 0.45 pg mL−1 (S/N = 3). It had good reproducibility (RSD = 1.90 %), repeatability (RSD = 2.6 %), and selectivity. After storing it at 4 ℃ for 15 days, its performance decreased to 92.37 %. And it had been successfully used to determine DCNP1 in the brain homogenate of mice with depression induced by chronic unpredictable mild stress (CUMS), providing a possible method for the rapid and accurate determination of DCNP1.

5-Alkyloxymethyl Derivatives of 2ʹ-Deoxyuridine Bearing 2,4-Dinitrophenyl and Dansyl Groups: Synthesis and Antibacterial Activity

Condensation of 3ʹ,5ʹ-di-O-acetyl-5-bromomethyl-2ʹ-deoxyuridine with 6-trifluoroacetylaminohexan1-ol yielded 5-(6-trifluoroacetylaminohexyl-1-oxymethyl)-3ʹ,5ʹ-di- O-acetyl-2ʹ-deoxyuridine. Its deblocking with an aqueous alcoholic solution of ammonia gave 5-(6-aminohexyl-1-oxymethyl)-2ʹ-deoxyuridine, and condensation with triazole and 2-chlorophenyl phosphorodichloridate followed by treatment with an aqueous solution of ammonia led to the formation of 5-(6-aminohexyl-1-oxymethyl)-2ʹ-deoxycytidine. The interaction of the obtained compounds with 2,4-dinitrofluorobenzene or N-hydroxysuccinimide esters of N-2,4-dinitrophenylaminohexanoic acid or 5-dimethylaminonaphthalene-1-sulfonyl glycine was used to synthesize DNP- and DNS-derivatives of 2ʹ-deoxyuridine as well as 5-(6-DNP-aminohexanoylaminohexyl-1-oxymethyl)-2ʹ-deoxycytidine. DNP derivatives of 2ʹ-deoxyuridine were shown to inhibit the growth of Micrococcus luteus.

Hydrogel Encapsulating Wormwood Essential Oil with Broad-spectrum Antibacterial and Immunomodulatory Properties for Infected Diabetic Wound Healing.

The integration of hydrogels with bio-friendly functional components through simple and efficient strategies to construct wound dressings with broad-spectrum antibacterial and immunomodulatory properties to promote the healing of infected diabetic wounds is highly desirable but remains a major challenge. Here, wormwood essential oil (WEO) is effectively encapsulated in the hydrogel via an O/W-Pickering emulsion during the polymerization of methacrylic anhydride gelatin (GelMA), acrylamide (AM), and acrylic acid N-hydroxysuccinimide ester (AAc-NHS) to form a multifunctional hydrogel dressing (HD-WEO). Compared with conventional emulsions, Pickering emulsions not only improve the encapsulation stability of the WEO, but also enhance the tensile and swelling properties of hydrogel. The synergistic interaction of WEO's diverse bioactive components provides a broad-spectrum antibacterial activity against S. aureus, E. coli, and MRSA. In addition, the HD-WEO can induce the polarization of macrophages from M1 to M2 phenotype. With these advantages, the broad-spectrum antibacterial and immunomodulatory HD-WEO effectively promotes the collagen deposition and neovascularization, thereby accelerating the healing of MRSA-infected diabetic wounds.

MPI/FLI/CTA multi-modality imaging of atherosclerotic plaque with plexind1-targeted nanoparticles

Abstract M1 macrophage is one of important components which contribute to the progression of atherosclerotic plaques.[1]Previous research of our team showed that plexind1 was expressed more in atherosclerotic plaques at vascular bifurcation than those at straight site and co-localized with M1 macrophages. This study aims to develop a novel multimodal imaging platform by designing a nanoprobe targeting plexind1 to detect and monitor bifurcation lesion and provide detecting method for animal research. Methods A novel multimodal imaging agent,A12-Fe3O4-Cy7, was constructed by coupling superparamagnetic iron oxide nanoparticle (SPIONs) with cyanine 7 N-hydroxysuccin imide ester（Cy7）and nanobody A12 which could target plexind1 specifically. The physical and biological properties were evaluated. To assess the A12-Fe3O4-Cy7 recognition and monitoring for plaque,high-fat-fed 10-week and 20-week Apoe-/- atherosclerosis mouse models were imaged using MPI and FLI,and both 3D images were fused with CTA. HE,Prussian blue and plexind1 immunohistochemical staining were performed on mouse carotid tissue sections. Results The A12-Fe3O4-Cy7 was of favorable stability, biocompatibility（concentration< 200ug/ml) and targeting effect.It could detect, locate, and monitor atherosclerotic plaques at bifurcation of carotid artery in ApoE-/- mice accurately and sensitively, showing significantly higher MPI and FLI signals both in vivo and ex vivo compared with both themselves before nanoprobe injection and two control groups(n=3,p<0.05). FLI is more sensitive than MPI to detect high-fat-fed 10-week mouse plaques.The results were confirmed histologically. Conclusion A12-Fe3O4-Cy7 might serve as a method to detect plaques at the bifurcation by targeting plexind1 in vivo. Limitations MPI is tested only in mouse models presently and it requires combination with CT to provide anatomical information. Besides，there was no intervention on plexind1,so the mechanism of plexind1 in bifurcation plaque occurrence and progression is not illustrated clearly,which is worth exploring in the future.In vitro and vivo assessmentIn vivo and ex vivo imaging

Molecular design of an ultra-strong tissue adhesive hydrogel with tunable multifunctionality.

Designing adhesive hydrogels with optimal properties for the treatment of injured tissues is challenging due to the tradeoff between material stiffness and toughness while maintaining adherence to wet tissue surfaces. In most cases, bioadhesives with improved mechanical strength often lack an appropriate elastic compliance, hindering their application for sealing soft, elastic, and dynamic tissues. Here, we present a novel strategy for engineering tissue adhesives in which molecular building blocks are manipulated to allow for precise control and optimization of the various aforementioned properties without any tradeoffs. To introduce tunable mechanical properties and robust tissue adhesion, the hydrogel network presents different modes of covalent and noncovalent interactions using N-hydroxysuccinimide ester (NHS) conjugated alginate (Alg-NHS), poly (ethylene glycol) diacrylate (PEGDA), tannic acid (TA), and Fe3+ ions. Through combining and tuning different molecular interactions and a variety of crosslinking mechanisms, we were able to design an extremely elastic (924%) and tough (4697kJ/m3) multifunctional hydrogel that could quickly adhere to wet tissue surfaces within 5s of gentle pressing and deform to support physiological tissue function over time under wet conditions. While Alg-NHS provides covalent bonding with the tissue surfaces, the catechol moieties of TA molecules synergistically adopt a mussel-inspired adhesive mechanism to establish robust adherence to the wet tissue. The strong adhesion of the engineered bioadhesive patch is showcased by its application to rabbit conjunctiva and porcine cornea. Meanwhile, the engineered bioadhesive demonstrated painless detachable characteristics and in vitro biocompatibility. Additionally, due to the molecular interactions between TA and Fe3+, antioxidant and antibacterial properties required to support the wound healing pathways were also highlighted. Overall, by tuning various molecular interactions, we were able to develop a single-hydrogel platform with an "all-in-one" multifunctionality that can address current challenges of engineering hydrogel-based bioadhesives for tissue repair and sealing.

Intracellular delivery of anticancer agents using dual responsive nanomicelles synthesized via RAFT polymerization

In this study, diselenide core crosslinked nanomicelles were developed for the delivery of docetaxel (DTX) to tumor cells, aiming to achieve synergistic antitumor chemotherapy in combination with radiotherapy.The nanomicelles were composed of a random triblock copolymer, Poly(poly(ethylene glycol) methyl ether methacrylate-co-N-isopropylacrylamide-co-Acrylic acid N-hydroxysuccinimide ester), synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and crosslinked using selenocystamine dihydrochloride. Physicochemical experiments were conducted to characterize the polymer synthesis and nanoparticles. The in vitro release profile was studied in response to redox conditions. In vitro cytotoxicity studies were performed on the breast cancer (MCF-7) cell line.The results showed the formation of 81 ± 5.2 nm spherical nanomicelles with a drug loading and encapsulation efficiency (EE) of 8.09% (w/w) and 97% (w/w), respectively. The nanomicelles exhibited dual responsive controlled release profiles, with approximately 80% of the drug being released within the first 2 h in response to intracellular glutathione (GSH) concentration and X-ray exposure. Fluorescent microscopy confirmed the effective internalization of the nanomicelles. Furthermore, the nanomicelles demonstrated increased cytotoxicity and apoptosis rates. The DTX-loaded nanomicelles exhibited a lower IC50 value (0.037 µg/ml) compared to free DTX (0.092 µg/ml) (P < 0.05). The combination of X-ray irradiation (2 Gy) and DTX release exhibited the highest cytotoxicity on MCF-7 cells (IC50 = 0.019 µg/ml).Overall, the nanomicelles prepared in this study, in combination with X-ray irradiation, demonstrated enhanced antitumor efficacy of the system.

Novel electrochemical biosensor for breast cancer detection, based on a nanocomposite of carbon nanofiber, metal–organic framework, and magnetic graphene oxide

In this study, we present the newly developed a novel microRNA biosensor based on magnetic rod carbon paste electrodes for breast cancer detection by using a relatively new MOF structure as a substrate. The major goal of manufacturing biosensors, suitable for clinical diagnostics, is to measure very low amount of microRNA 155 in complex environments. Therefore, we used a combination of different materials, including carbon nanofibers, CuBTC-AIA (CuMOF), and Fe@rGO, to improve the electrode surface-to-volume ratio and facilitate the electron transfer process. In this method, 1-pyrenebutyric acid N-hydroxysuccinimide ester was used to bind the microRNAs to the electrode surface. The hybridization process on the modified electrode surface was investigated using cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry across the potential range, in which the accumulated hematoxylin was electroactive. Under optimal conditions, a very low detection limit of 0.08 fM and an adequate dynamic range of 0.2 fM–500 pM were achieved. The fabricated sensor was reported to be reproducible and selective when tested using different types of mismatched target sequences. And finally, the real human serum samples were used to confirm the capability of the nanobiosensor to detect microRNA 155 without any significant interference from other molecules and components.

Specific Targeting of STAT3 in B Cells Suppresses Progression of B Cell Lymphoma.

The signal transducer and activator of transcription 3 (STAT3), which regulates multiple oncogenic processes, has been found to be constitutively activated in lymphoma, suggesting its potential as a therapeutic target. Here, we constructed an anti-CD19-N-(4-carboxycyclohexylmethyl) maleimide N-hydroxysuccinimide ester (SMCC)-protamine (CSP)-STAT3 small interfering RNA (siRNA) conjugate and demonstrated that the CSP-STAT3 siRNA conjugate could specifically bind to normal B cells and A20 lymphoma cells in vitro. It decreased the STAT3 expression in B cell lymphoma cell lines (A20, SU-DHL-2 and OCI-Ly3), resulting in reduced proliferation of lymphoma cells featured with lower S-phase and higher apoptosis. Using an A20 transplantable lymphoma model, we found that the CSP-STAT3 siRNA conjugate significantly inhibited tumor growth and weight. Ki-67, p-STAT3, STAT3, and serum IL-6 levels were all significantly reduced in A20-bearing mice treated with CSP-STAT3 siRNA. These findings indicate that specifically targeting STAT3 siRNA to B cell lymphoma cell lines can significantly decrease STAT3 activity and inhibit tumor progression in vitro and in vivo, suggesting its potential utilization for cancer treatment.

In vivo fluorescent labeling and tracking of extracellular matrix.

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.

Facile immobilization of pyridoxal 5′-phosphate using p-diazobenzoyl-derivatized Sepharose 4B

Pyridoxal 5′-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ∼2.6 µmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors.

Tissue-Adhesive Hydrogel Spray System for Live Cell Immobilization on Biological Surfaces.

Gelatin hydrogels are used as three-dimensional cell scaffolds and can be prepared using various methods. One widely accepted approach involves crosslinking gelatin amino groups with poly(ethylene glycol) (PEG) modified with N-hydroxysuccinimide ester (PEG-NHS). This method enables the encapsulation of live cells within the hydrogels and also facilitates the adhesion of the hydrogel to biological tissues by crosslinking their surface amino groups. Consequently, these hydrogels are valuable tools for immobilizing cells that secrete beneficial substances in vivo. However, the application of gelatin hydrogels is limited due to the requirement for several minutes to solidify under conditions of neutral pH and polymer concentrations suitable for live cells. This limitation makes it impractical for use with biological tissues, which have complex shapes or inclined surfaces, restricting its application to semi-closed spaces. In this study, we propose a tissue-adhesive hydrogel that can be sprayed and immobilized with live cells on biological tissue surfaces. This hydrogel system combines two components: (1) gelatin/PEG-NHS hydrogels and (2) instantaneously solidifying PEG hydrogels. The sprayed hydrogel solidified within 5 s after dispensing while maintaining the adhesive properties of the PEG-NHS component. The resulting hydrogels exhibited protein permeability, and the viability of encapsulated human mesenchymal stem/stromal cells (hMSCs) remained above 90% for at least 7 days. This developed hydrogel system represents a promising approach for immobilizing live cells on tissue surfaces with complex shapes.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Front Cover: Unique Crosslinking Properties of Psoralen‐Conjugated Oligonucleotides Developed by Novel Psoralen N‐Hydroxysuccinimide Esters (ChemBioChem 15/2023)

Psoralens and their derivatives have unique crosslinking features to DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable. In this study, we developed novel psoralen N-hydroxysuccinimide esters that allow the introduction of psoralens into any amino-modified oligonucleotides. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools. More information can be found in the Research Article by A. Yamayoshi et al.

Cartilage Lacuna-Inspired Microcarriers Drive Hyaline Neocartilage Regeneration.

Cartilage equivalents from hydrogels containing chondrocytes exhibit excellent potential in hyaline cartilage regeneration, yet current approaches have limited success at reconstituting the architecture to culture nondifferentiated chondrocytes in vitro. In this study, specially designed lacunar hyaluronic acid microcarriers (LHAMCs) with mechanotransductive conditions that rapidly form stable hyaluronic acid (HA) N-hydroxy succinimide ester (NHS-ester) are reported. Specifically, carboxyl-functionalized HA is linked to collagen type I via amide-crosslinking, and gas foaming produced by ammonium bicarbonate forms concave surface of the microcarriers. The temporal 3D culture of chondrocytes on LHAMCs uniquely remodels the extracellular matrix to induce hyaline cartilaginous microtissue regeneration and prevents an anaerobic-to-aerobic metabolism transition in response to the geometric constraints. Furthermore, by inhibiting the canonical Wnt pathway, LHAMCs prevent β-catenin translocation to the nucleus, repressing chondrocyte dedifferentiation. Additionally, the subcutaneous implantation model indicates that LHAMCs display favorable cytocompatibility and drive robust hyaline chondrocyte-derived neocartilage formation. These findings reveal a novel strategy for regulating chondrocyte dedifferentiation. The current study paves the way for a better understanding of geometrical insight clues into mechanotransduction interaction in regulating cell fate, opening new avenues for advancing tissue engineering.

Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.