Bioconjugate Chemistry

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase(R) platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a "What You See Is What You Get" (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines.

Recombinant peptide production was pioneered in the 1970s for the generation of therapeutic peptides, with notable examples including insulin and somatostatin. These early methods required the use of cyanogen bromide (BrCN) for cleavage of the native peptide sequence from a fusion protein. Since that time, while numerous BrCN-dependent peptide methods continue to be reported, the accessibility and cost of site-specific proteases have improved dramatically. These developments have enabled alternative approaches to recombinant peptide generation that obviate the need for BrCN, an environmentally destructive toxin. We recently created an immobilized SUMO protease that can replace BrCN usage in recombinant peptide production workflows by releasing native peptides expressed as part of a SUMO-peptide fusion protein. We have used this approach to generate P113 peptide, the minimal active fragment of the antifungal peptide Histatin 5. In this report, we describe the creation and characterization of this immobilized SUMO protease and its application in the production of experimentally viable quantities of active P113 peptide.

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC), which are responsible for severe foodborne infections that can progress to hemolytic-uremic syndrome (HUS). Currently, no specific anti-toxin therapies are available. In this study, we devised a glycoengineering strategy utilizing Functional-Spacer-Lipid (FSL) conjugates to create small extracellular vesicles (sEVs)-based decoy receptors for Shiga toxin type 1 (Stx1). sEVs isolated from human Caco-2 and HEK293T cells were functionalized with Gb3 trisaccharide (Gal1[->]4Gal{beta}1[->]4Glc)-containing FSL conjugates, yielding Gb3-decorated vesicles, displaying the Gal1[->]4Gal epitope. Characterization of FSL-modified sEVs confirmed that FSL incorporation did not adversely affect sEV morphology, size distribution, or surface charge. Western blotting and bead-assisted flow cytometry verified the presence of exosomal markers (CD9 and CD63) and the Gb3 epitope on modified vesicles. Gb3-tagged sEVs from both cell types exhibited high specificity in binding Stx1B, while control vesicles carrying Galili epitope (Gal1[->]3Gal{beta}1[->]4GlcNAc), lacking Stx1B binding, demonstrated negligible binding. Gb3-expressing Caco-2 cell-based assays revealed that Gb3-decorated sEVs markedly reduced Stx1B binding to Caco-2 cells, indicating effective competition with cellular receptors. Furthermore, glycoengineered sEVs did not impair Caco-2 cell viability at concentrations sufficient for Stx1B sequestration. These findings establish FSL-mediated glycoengineering as a rapid and versatile approach for generating sEV-based decoy receptors that effectively bind Stx1B. Gb3-containing human sEVs may serve as an agent for neutralizing Stx1B and potentially other glycan-binding toxins, supporting the development of promising next-generation anti-toxin therapeutics.

Non-invasive tracking of natural killer (NK) cells remains a major challenge in cancer immunotherapy, limiting our understanding of their in vivo migration and persistence. Magnetic particle imaging (MPI) offers a quantitative, real-time method for visualizing labeled cells, yet optimal labeling protocols for NK cells have not been established. Here, we evaluate commercially available iron oxide nanoparticles (IONPs) for MPI labeling of both NK92MI cells and primary human NK cells. Labeled cells retained viability and cytotoxicity, including activity against three-dimensional tumor spheroids, and were detectable by MPI. To further examine imaging performance in a biologically relevant context, we employed mouse phantoms that recapitulate organ-specific signal distributions, enabling evaluation of quantification and liver spillover effects. We identify key tradeoffs between particle colloidal stability and per-cell iron content: VivoTrax and VivoTrax Plus provided higher MPI signal but required post-labeling purification, reducing cell recovery, whereas Synomag-D and Perimag were more stable and preserved cell yield despite lower signal intensity per cell. These results provide a framework for selecting nanoparticles that balance detection sensitivity, cell viability, and workflow practicality, advancing non-invasive NK cell tracking.

Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors including small cell lung cancer (SCLC) and represents a validated target for peptide receptor radionuclide therapy. The SSTR2 agonist [177Lu]Lu-DOTA-TATE is clinically approved, however, treatment resistance and relapse occur. The SSTR2 antagonist SSO110 (DOTA-JR11, OPS201) demonstrates higher tumor uptake and longer retention than DOTA-TATE both pre-clinically and clinically. We performed a systemic head-to-head comparison of SSO110 labeled with various radionuclides of distinct emission characteristics to identify the optimal radionuclide for SSO110 and to compare antagonist with agonist performance. MethodsSSO110 was radiolabeled with 177Lu, 161Tb, 212Pb, and 225Ac. Biodistribution was assessed in AR42J and NCI-H69 xenograft models. Therapeutic efficacy of single and fractionated [212Pb]Pb-SSO110 was compared with [177Lu]Lu-SSO110 in NCI-H69 tumors. Single-dose efficacy of 225Ac-, 161Tb-, and 177Lu-labeled SSO110 was evaluated in both models. [{superscript 2}{superscript 2}Ac]Ac-DOTA-TATE served as agonist comparator. Tumor growth, survival, safety parameters, and tumor absorbed doses were analyzed. ResultsAll SSO110 radioconjugates demonstrated comparable biodistribution with high tumor uptake and favorable tumor-to-kidney ratios. In NCI-H69 tumors, [212Pb]Pb-SSO110 induced dose-dependent tumor growth delay but did not improve anti-tumor efficacy compared with [177Lu]u-SSO110 under single or fractionated regimens. [161Tb]Tb-SSO110 showed efficacy comparable to [177Lu]Lu-SSO110 in NCI-H69 model and significantly improved tumor growth delay in high-SSTR2-expressing AR42J tumors. Across both models, [225Ac]Ac-SSO110 demonstrated the highest therapeutic potency, inducing durable tumor regression and 100% survival at clinically relevant activities. [225Ac]Ac-SSO110 also outperformed the agonist comparator [225Ac]Ac-DOTA-TATE. Dosimetry analysis revealed a 63-fold higher tumor absorbed dose per injected administered activity for [225Ac]Ac-SSO110 compared with [212Pb]Pb-SSO110. All treatments were well tolerated without significant renal or hepatic toxicity. ConclusionTherapeutic efficacy of SSTR2-targeted peptide receptor radionuclide therapy appears to benefit from alignment between radionuclide physical half-life and ligand tumor residence time. Among the radionuclides evaluated, [225Ac]Ac-SSO110 demonstrated the most pronounced and durable anti-tumor efficacy, outperforming [161Tb]Tb-SSO110, [177Lu]Lu-SSO110, and the short-lived -emitter [212Pb]Pb-SSO110. These findings support clinical investigation of [225Ac]Ac-SSO110 in SSTR2-positive malignancies.

Genetically encoded calcium ion (Ca2+) indicators (GECIs) enable visualization of Ca2+ dynamics in living systems but often suffer from limited photostability during prolonged imaging. The recent discovery of StayGold, a green fluorescent protein (FP) with exceptional brightness and photostability, opened the possibility of addressing this longstanding challenge. Here, we sought to establish whether a monomeric variant of StayGold (mStayGold) could be converted into a single FP-based GECI. Through extensive protein engineering, we generated a functional mStayGold-based GECI, HiCaRI (Highly intensiometric Ca2+ Responsive Indicator) by fusing Calmodulin (CaM) and the ckkap binding peptide from K-GECO1 into mStayGold(J). HiCaRI exhibits a large Ca2+-dependent inverse fluorescence response ({Delta}F/Fmin = -15) while retaining high brightness and improved photostability relative to previously reported GFP-based GECIs. Although the current variant represents a first-generation prototype with shortcomings in terms of Ca2+ affinity and photostability (relative to StayGold and mStayGold(J)), this work demonstrates the feasibility of constructing single FP-based GECIs from a highly photostable fluorescent protein.

{beta}4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit {beta}4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 {beta}-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked {beta}3-galactosylation towards mAb Fc N-linked {beta}4-galactosylation. The second event overexpresses {beta}-1,4-galactosyltransferase 1 ({beta}4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc {beta}4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and {beta}4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout attenuates UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans. HighlightsO_LIDual COSMC KO and {beta}4GalT1 overexpression achieves >90% mAb Fc galactosylation. C_LIO_LICOSMC KO redirects UDP-Gal from O-glycans to mAb Fc without impacting cell growth. C_LIO_LIDual glycoengineering reduces production of undesired Man5 glycans. C_LI

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within minutes at nanomolar substrate concentrations, and can be multiplexed with other self-labeling tags such as SNAP-tag2 and HaloTag7. These advances establish CLIP-tag2 as a powerful tagging platform for high-performance live-cell bioimaging.

Poly(ADP-ribose) polymerase 1 (PARP1) is a central mediator of DNA damage repair and an established therapeutic target in homologous recombination-deficient cancers. Radiolabelled PARP inhibitors provide a strategy to deliver cytotoxic radiation directly to tumour DNA by exploiting PARP overexpression and trapping at sites of DNA damage. Here, we describe the design, radiosynthesis, and in vitro evaluation of [123I]Italia, a talazoparib-derived Auger electron-emitting agent for PARP-targeted radionuclide therapy. Stereochemically pure [123I]Italia, (8S,9R)-5-fluoro-8-(4-(iodo-123I)phenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one was synthesised in one step via copper-mediated iodo-deboronation, achieving activity yields >80% and molar activities >6.2 {+/-} 3.1 GBq/{micro}mol (n=8). UPLC analysis confirmed radiochemical purity >97%. Italia exhibited potent PARP1 inhibition (IC50 0.48 nM) and in silico predicted binding affinity comparable to talazoparib. In a panel of PARP-expressing cancer cell lines, [123I]Italia demonstrated highest uptake at 60 min, PARP-selective uptake, predominant nuclear localisation (up to 60% of added activity) and chromatin association consistent with PARP trapping (up to 15% of total activity recorded). Uptake was reduced more than 50-fold by addition of an excess of any PARP inhibitor (e.g. olaparib, talazoparib, and rucaparib) and in PARP1 knockout cells, confirming target specificity. Clonogenic assays showed a marked, added activity-dependent reduction in survival of PARP-expressing cells following a brief one-hour exposure, whereas PARP1-deficient cells were resistant. Collectively, these findings identify [123I]Italia as a promising PARP-targeted Auger electron-emitting theranostic candidate that warrants further in vivo evaluation.

Fluorescent proteins (FPs) are essential tools for biological imaging but are limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. Despite extensive use, the molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decay in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, and fluorescence decay during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching strongly depends on FP. It involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON-OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.

Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However, its reliance on hazardous organic solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid-phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here, we demonstrate that industrially relevant 2-chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate-free 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC{middle dot}HCl) and Oxyma activation system with a synergistic thermal-acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel-forming peptides, opioid peptides derived from enkephalins, toxin-inspired sequences, and a lipid-interacting fragment of -synuclein. Analytical characterization by HPLC and MALDI-TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal-acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.

Spatiotemporal regulation of mRNA translation is central to gene expression. Over the past decade, translation has become directly observable in live cells at single-mRNA resolution by tagging nascent chains with tandem arrays of short epitope tags recognized by genetically encodable fluorescent intracellular antibodies (intrabodies). While this technology has revolutionized our understanding of translation regulation, the current toolbox of tagging systems remains limited. Here, we developed a novel and tight-binding intrabody against a short (11-amino acid) HIV protease epitope (named UTag). To ensure robust intracellular folding of the anti-UTag intrabody, we further engineered a cysteine-free variant that folds and functions independently of disulfide-bond formation, as validated by X-ray crystallography. The cysteine-free anti-UTag intrabody retains high binding affinity comparable to the parental intrabody while exhibiting significantly improved thermostability ([~]80 {degrees}C). Importantly, the cysteine-free UTag system enables real-time tracking of single-mRNA translation in live cells with performance on par with the parental UTag system as well as the established SunTag and ALFA-tag. Collectively, these results demonstrate that the newly developed UTag system expands the toolbox for live-cell translation tracking and provides complementary tools for multiplexed applications.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

It has been hypothesized that effective cellular internalization is required for the retention of 225Ac daughter radionuclides. The complex decay chain of 225Ac and recoil-mediated release of daughters, particularly 213Bi (half-life (t1/2) = 46 min), raise concerns about redistribution that may reduce tumor absorbed dose (TAD) and increase off-target radiation exposure. Because somatostatin receptor subtype 2 (SSTR2) antagonists such as SSO110 are not internalized, it has been proposed that the daughter radionuclides are less effectively retained compared to internalizing agonists such as DOTA-TATE. We therefore performed a direct and quantitative comparison of daughter radionuclide redistribution following administration of [225Ac]Ac-SSO110 and [225Ac]Ac-DOTA-TATE. MethodsBiodistribution and 213Bi redistribution were evaluated in Balb/c nude mice bearing NCI-H69 small cell lung cancer xenografts. Repeated gamma counting combined with bi-exponential modeling was used to quantify 225Ac and 213Bi activity in tumor, blood, bone marrow, kidneys, liver, and intestines up to 96 h post-injection. TAD was calculated with and without accounting for experimentally-derived 213Bi redistribution. Real-time in vitro binding assays were conducted to characterize cellular retention of [225Ac]Ac-SSO110. Results[225Ac]Ac-SSO110 demonstrated higher tumor uptake and prolonged retention compared with [225Ac]Ac-DOTA-TATE, resulting in a 1.9-fold higher tumor-to-kidney ratio at 96 h and a 2.8-fold higher TAD. Redistribution of 213Bi from tumor was minimal and comparable between agonist and antagonist, with maximum tumor loss of 3.5% for [225Ac]Ac-SSO110 and 2% for [225Ac]Ac-DOTA-TATE. Accounting for daughter redistribution reduced TAD by less than 5% for both radioconjugates. No sustained 213Bi accumulation was observed in blood, kidneys, or liver, and only minimal activity was detected in bone marrow and intestines. Real-time binding studies demonstrated sustained cell-associated {beta}- signal following incubation with [225Ac]Ac-SSO110. ConclusionReceptor-mediated internalization is not required for effective retention of 225Ac daughter radionuclides. Despite negligible internalization, [225Ac]Ac-SSO110 achieved superior TAD and higher tumor-to-kidney ratio without increased daughter redistribution compared with the internalizing agonist [225Ac]Ac-DOTA-TATE. These findings question the necessity of internalization for daughter retention and support further evaluation of antagonist-based 225Ac radioligand therapy.