The emerging field of glycoRNAs, RNA molecules covalently modified with glycans, challenges the long-held belief that glycosylation is exclusive to proteins and lipids. The discovery of 3-(3-amino-3-carboxypropyl) uridine (acp3U) as a specific N-glycan attachment site has been a major breakthrough, establishing glycoRNA as a structurally defined and functionally relevant biopolymer. This new function of acp3U suggests its crucial regulatory node that correlates translation with other cellular processes, such as immune modulation and cell signaling. The presence of glycoRNAs on the cell surface and their interaction with immune receptors imply their involvement in cell-to-cell communication. Furthermore, studies have begun to associate altered glycoRNA patterns with conditions like cancer and inflammation, opening up possibilities for diagnostic and therapeutic applications. Despite the rapid progress in this field, several key challenges remain, including the inherent bias of current detection methods, the difficulty of isolating pure glycoRNA samples from complex cellular mixtures, and the largely unknown mechanisms of specific glycan linkages. Future research must focus on developing unbiased and sensitive analytical technologies to accurately map these modification patterns at a single-nucleotide resolution. This review summarizes the chemical and enzymatic mechanisms of RNA glycosylation sites, highlights its potential functional roles in cells, and outlines future research aimed at uncovering its full biological and therapeutic potential.

In vitro transcription (IVT) using T7 RNA polymerase is a key step in mRNA synthesis for therapeutic applications. However, the generation of double-stranded RNA (dsRNA) byproducts during IVT─primarily due to RNA rebinding and self-priming─triggers innate immune responses and reduces translation efficiency. Here, we present a simple and effective strategy to minimize dsRNA formation during IVT by incorporating PEGylated graphene oxide (PEG-GO). Graphene oxide (GO) preferentially binds single-stranded nucleic acids, but its use is limited by protein adsorption and low solubility in Mg2+-containing buffers. PEG modification improves GO's dispersibility and reduces protein binding, allowing selective sequestration of nascent RNA without inhibiting T7 RNA polymerase activity. The addition of PEG-GO to the IVT reaction reduced the dsRNA content by over 75% while maintaining RNA yield and accelerating transcription kinetics. Moreover, mRNA synthesized in the presence of PEG-GO showed enhanced protein expression and reduced interferon-β secretion in transfected cells, comparable to post-IVT-purified mRNA. Our work demonstrates PEG-GO as a practical additive for improving the quality and scalability of IVT-based mRNA production.

The scarcity of a simple, cost-effective, and green method for the immobilization of enzymes severely hampers their application. Herein, a versatile and mild xylanase and lichenase bienzyme (XLBE) immobilization strategy including biofunctionalization of the magnetic particles, enzyme-free purification, and spontaneous covalent bridging based on SpyCatcher "Click Biology" was proposed. Only biocompatible tannic acid (TA), Fe3+, and elastin-like polypeptide-SpyCatcher were fed. The biomodified magnetic particles exhibited excellent stability with a loss of only 3.51% EC after 1 h of incubation at pH 7.5. Then, they were applied to immobilize SpyTag fused XLBE directly from the crude solution at a loading of 12.5 mg/g. The retention of XLBE and the xylanase activity were as high as 87.73% and 82.77%, respectively. The half-lives of the immobilized xylanase increased by 1535.75% (50 °C) compared to those of the free xylanase. The immobilized XLBE showed excellent reusability, retaining 70.15% (xylanase) and 78.81% (lichenase) of the initial activity after 8 cycles of recycling. They also showed superior catalytic performance with 202.25% improvement in green production of total reducing sugar and 30.77% improvement in juice clarification. Moreover, the versatility of the immobilization strategy was also demonstrated on inorganic carriers such as silicon dioxide and carbon nanotubes. This innovative all-in-one strategy avoids too many chemical reagents for surface modification and omits the complex enzyme prepurification process for immobilization, which will shed light on the green biocatalytic applications based on time-effective and low-byproduct surface functionalization strategies.

Red blood cells (RBCs) have been employed to convey and deliver a variety of therapeutic agents, from small molecules to proteins. The therapeutics are typically installed within the RBC interior via a pore-forming process that results in membrane disruption and a partial loss of hemoglobin. An alternative approach, namely appending therapeutics to the RBC surface, has received significantly less attention. Here we focus on the characterization of an array of membrane anchoring modalities (noncovalent, reversible covalent, and covalent). Surface modification is experimentally simpler and structurally less invasive than its membrane disruptive counterpart. This panel is designed, synthesized and assessed with respect to RBC loading capacity, retention, and rate of transfer to other cell populations. The cell surface anchors are appended to a structural scaffold (cobalamin) that can house and deliver therapeutic agents. Imaging studies for a series of representative derivatives reveal that these species are not internalized by the RBCs, consistent with the absence of an active endocytic pathway in mature RBCs. Furthermore, enzymatic digestion of the glycocalyx failed to impair loading or retention, suggesting that the derivatives are likely anchored to the RBC membrane. The structural motifs identified in this study provide a template for the development of membrane tethered therapeutics that are specifically designed to be transported to diseased sites by RBCs.

Therapeutic mRNA has received significant attention as a new class of nucleic acid-based medicine due to its promising potential toward protein replacement therapy, vaccine development, and genome editing. Unlike DNA-based therapies, which depend on nuclear entry, mRNA works directly in the cytoplasm to produce proteins. Nevertheless, the delivery of large nucleic acids, such as mRNA, remains an unresolved challenge due to their instability, limited cellular uptake, and the cytotoxicity commonly associated with several cationic carriers. In recent years, various platforms have been developed for delivering mRNAs, including lipid nanoparticles, liposomes, dendrimers, and polyion complex micelles. Despite their success, each of these platforms faces important challenges, such as cytotoxicity, poor encapsulation efficiency and stability, limited endosomal escape, and reduced effectiveness in biological media. As a general alternative, in this study, we developed a peptide-based artificial viral capsid modified with a cell-penetrating peptide possessing perfluoroalkyl (PFA) chain (CADC6F13F) for the efficient and safe delivery of mRNAs into cells. The PFA-modified artificial viral capsid was formed by the self-assembly of the CADC6F13F-conjugated β-annulus peptide, unmodified β-annulus, and dT20-SS-β-annulus, which can be hybridized with the poly(A) tail of the mRNA. Dynamic light scattering and transmission electron microscopy confirmed the formation of mRNA-encapsulated spherical capsids of approximately 200 nm in diameter. Importantly, PFA modification of the artificial viral capsid significantly improved the delivery efficiency and minimized cytotoxic effects. Fluorescence images further demonstrated that cells treated with these capsids exhibited significantly higher expression of mCherry-encoding mRNAs, indicating successful delivery and translation. Overall, our study introduces a promising viral-mimetic approach for mRNA therapeutics without compromising safety and efficiency.

Breast cancer remains a leading cause of cancer-related death worldwide, partly due to disease heterogeneity and the lack of reliable biomarkers. The G protein-coupled oxytocin receptor (OTR) has emerged as a potential biomarker and therapeutic target in breast cancer, as its overexpression correlates with tumor growth and metastasis. OTR thus presents new opportunities for molecular imaging and targeted therapy in breast cancer. This study explores three novel 68Ga-labeled peptides as potential OTR-specific imaging agents. Their preclinical evaluation includes in vitro assays and positron emission tomography (PET) studies in breast cancer models. The work also introduces the application of linchpin chemistry with LP1 and LP2 as a novel strategy for attaching bifunctional chelating agents. This tandem approach not only enables efficient peptide cyclization but also facilitates radiometal incorporation, representing a versatile platform for the design of next-generation radiopharmaceuticals. Binding studies using an aequorin-based assay in CHO cells expressing human OTR revealed the following EC50 values: natGa-LP1-oxytocin (376 nM), natGa-DOTA-Lys8-oxytocin (1.38 nM), and natGa-LP2-oxytocin (123 nM). Radiolabeling with 68Ga was efficient and reproducible, consistently yielding high decay-corrected radiochemical yields of 52-74% and high radiochemical purity >98%. PET imaging demonstrated maximum MCF-7 tumor uptake for 68Ga-LP1-oxytocin (SUVmax 0.64 ± 0.10; n = 3) and 68Ga-LP2-oxytocin (SUVmax 0.64 ± 0.05; n = 7) at 10 min postinjection, whereas 68Ga-DOTA-Lys8-oxytocin reached comparable uptake (SUVmax 0.64 ± 0.12; n = 3) at 30 min. Notably, 68Ga-LP2-oxytocin showed superior background clearance and faster blood pool washout. Tumor uptake specificity was verified through competitive inhibition studies: predosing with oxytocin reduced tracer accumulation in a concentration-dependent manner at 10 min postinjection, with decreases of 33% at 50 μM and 68% at 300 μM, confirming selective OTR-mediated binding in vivo. Among the evaluated tracers, the novel 68Ga-LP2-oxytocin peptide demonstrated efficient radiolabeling, strong binding potency, and favorable in vivo characteristics, including uptake in estrogen receptor-positive MCF-7 tumors and superior background and clearance profiles. With further structural optimization, 68Ga-LP2-oxytocin holds promise as a PET radioligand for targeting OTR in breast cancer.

An affinity-guided photo-cross-linking reaction based on Fc-binding peptide harboring p-benzoyl-l-phenylalanine (PEptide-DIrected Photo-cross-linking; PEDIP) enables site-specific modification of native antibodies but suffers from issues coming from long UV exposure and high peptide concentrations. In this study, we report a bacterial surface-display system of the photo-cross-linking peptide and high-throughput screening of its libraries with FACS for higher photo-cross-linking efficiency. The lead peptide (B1) exhibited a higher conjugation yield than the original peptide (95.5% vs 78.4%) while preserving site fidelity at heavy chain Met252, confirmed by LC-MS/MS. A cyclooctyne group introduced to the N-terminus of B1 enabled conjugation of IgG with payloads via strain-promoted azide-alkyne cycloaddition. The conjugate of trastuzumab (antihuman HER2 IgG) and monomethyl auristatin retained antigen selectivity and exhibited potent cytotoxicity in HER2+ HCC1954 cells with minimal activity in HER2- MDA-MB-231 cells.

Precise glioma detection is a critical challenge in the clinic. Magnetic particle imaging (MPI) is an emerging, highly sensitive medical imaging technique that has the potential to accurately detect glioma at the molecular and cellular levels. Magnetic nanoparticles (MNPs) provide an effective approach for targeted imaging to specific regions, and the morphology of MNPs plays a vital role in determining their MPI performance. MNPs with various shapes have been developed to pursue sensitive MPI, while the effect of the multivoid structure on MPI tracers is still unrevealed. Herein, we systematically investigate the impact of multivoid, yolk-shell, and completely hollow structures on the MPI signal. We identify that an increased number of magnetic cores per unit volume, decreased coercivity, and reduced full width at half-maximum of the magnetization derivative caused by the multivoid structure are the key factors that endow tracers with high MPI sensitivity. Moreover, further Arginine-Glycine-Aspartic Acid peptide modification ensures that the multivoid nanotracer exhibits high affinity and targeting to tumor cells and tissues, providing an obvious MPI signal to achieve precise glioma detection. This work enables a fundamental understanding of the effect of the multivoid structure on the MPI signal, lending guidance for designing high-performance MPI tracers for biomedical applications and promoting precise disease diagnosis.

DNA topoisomerase I (TOP1) inhibitor-based antibody-drug conjugates (ADCs) incorporating photosensitive camptothecin (CPT) analogs as payloads have emerged as a promising therapeutic strategy in oncology. However, their clinical potential is challenged by photoinduced instability during manufacturing, storage, and handling, which are typically conducted under ambient light conditions, using white light with wavelengths greater than 400 nm and minimal ultraviolet (UV) exposure. In this study, we systematically investigated, for the first time, the impact of ambient light exposure on TOP1 inhibitor-conjugated ADCs (TOP1-ADCs), and we revealed critical photodegradation mechanisms that compromise their physicochemical properties and therapeutic efficacy. Upon ambient light exposure, TOP1-ADCs underwent significant chemical, physical, and biofunctional changes, including visible color changes, aggregation, oxidation, drug loss, payload degradation, destabilization in CH2 domain, and reduced binding affinity to the neonatal Fc receptor (FcRn). Mechanistic studies revealed two distinct pathways driving this photodegradation: a reactive oxygen species (ROS) generation-mediated pathway and a direct payload self-photolysis-mediated pathway. In oxygen-rich environments, the ROS-generation-mediated pathway predominates, where the excited-state payload primarily transfers energy to molecule oxygen to induce ROS formation, leading to oxidation and subsequent aggregation and drug loss. Under oxygen-depleted conditions, direct payload photolysis becomes the primary degradation mechanism, resulting in payload degradation and more severe particular nonreducible aggregation formation. These findings highlighted the necessity of implementing stringent light-protective measures throughout the production, storage, and handling of TOP1-ADCs to preserve their stability, efficacy, and safety. The study provided critical insights into the photosensitivity of TOP1 inhibitor-based ADCs, offering a foundation for optimizing their development and clinical applications.

Multiple sclerosis (MS) is an autoimmune disease that causes neural degeneration as a result of the immune system launching an attack on the myelin sheath surrounding neurons. MS has multiple disease states; each one has been associated with a different onset pathway and requires a separate treatment. Primary progressive MS (PPMS) is a rare form of MS that affects 10-15% of MS patients, and Ocrelizumab is currently the only FDA-approved treatment on the market. While it can be effective in managing PPMS, Ocrelizumab can only delay the onset of the disease. In this study, MOG-Fc-BPI was designed as a potential therapeutic agent to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner, altering immune cells from an inflammatory to a regulatory phenotype. Here, MOG-Fc-BPI was successfully synthesized by conjugating the MOG-R5 peptide using sortase A enzyme to the C-terminus of the Fc-domain with LABL peptide at the N-terminus. Purified MOG-Fc-BPI was formulated to reach a concentration of 15 mg/mL for the in vivo study. MOG-stimulated EAE in C57BL/6 mice (a model for PPMS) that were treated with MOG-Fc-BPI on days 4 and 7 at 35 nmol/dose showed complete disease suppression on day 19 (score = 0; without symptoms) compared to PBS. The MOG-Fc-BPI-treated mice showed increased body weights throughout the study, while PBS-treated mice lost around 10% bodyweight during the peak of the disease without recovery up to the end of the study. Overall, this study provided a proof-of-concept that MOG-Fc-BPI has the potential to suppress PPMS.