Bioconjugate Chemistry

Glioblastoma (GBM) is the most common primary malignant brain tumor and is typically fatal. GBM therapies are hindered by the impermeability of the blood brain barrier (BBB), the diffuse and infiltrative nature of the tumor, and the high heterogeneity of intratumoral GBM cells. Aptamers are short, synthetic, folded single strands of RNA or DNA or analogs that bind targets with high affinity and specificity. Aptamers are developed via the principles of natural selection, permitting an unbiased approach to therapeutic development. Thus, rather than using rational design to select a target and develop a targeting moiety, cycles of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are employed in cell culture or in vivo to identify aptamers against unknown targets. Antibody drug conjugates (ADCs) have shown some efficacy for GBM but are limited by their large size and thus depend on leakiness of the BBB. We have recently applied in vivo SELEX to develop anti-GBM aptamers (six-fold smaller in mass than IgG antibodies) and to select aptamer-drug conjugates. Here we report attempts to focus aptamer selection toward internalizing drug-delivery targets and resulting challenges involving loss of tumor specificity in vivo.

Fluorescence imaging in the second near-infrared window (NIR-II, 950-1700 nm) offers reduced scattering, lower autofluorescence, and deeper tissue penetration than NIR-I imaging, but its adoption is limited by the need for custom-synthesized fluorophores. Here, we identify commercially available dyes that exhibit usable NIR-II emission. Eleven visible, far-red, and NIR-I fluorophores were screened under twelve acquisition configurations combining 670, 760, and 808 nm excitation with band-pass (950 nm, 1400 nm) or long-pass (1000 nm, 1250 nm) emission filters. Output varied markedly with fluorophore identity and excitation/emission configuration. Among hydrophobic dyes, DiR exhibited strong emission across almost all excitation and emission filters. Among hydrophilic dyes, strong NIR-II fluorescence was observed for IRDye 680RD (excitation at 670 nm), sulfo-cyanine 7 (excitation at 670 nm and 760 nm), and indocyanine green (excitation at 808 nm). DiR showed a linear concentration-response under 760 nm excitation with BP1400 detection. Upon encapsulation in PEGylated liposomes, strong NIR-II fluorescence was retained. In an in vivo study in mice, NIR-II resolved vasculature that NIR-I could not consistently delineate, and enabled pharmacokinetic analysis. Both windows returned similar ex vivo organ distributions. NIR-II imaging is therefore accessible using commercial off-the-shelf fluorophores, provided the dye is matched to the intended excitation/emission configuration.

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

Rationale: Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods: Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidaks multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111In-Dinutuximab-IRDye800. Results: Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 microgram dose of 111In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions: 111In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

Targeted radiopharmaceutical development for ovarian cancer (OC) has been limited by the lack of molecular targets that combine broad tumor expression with minimal normal-tissue distribution. TRA-1-60 (TRA) is a cancer-associated glycoepitope carried by podocalyxin. Here, we evaluated TRA as a target for OC and developed a TRA-directed immunoPET imaging platform. Immunohistochemical analysis demonstrated significantly higher TRA expression in ovarian tumors than in normal adjacent ovarian tissue, with expression maintained across epithelial OC histotypes and disease stages. An engineered anti-TRA single-chain variable fragment-Fc (scFv-Fc) demonstrated robust penetration of three-dimensional tumor spheroids and selective accumulation in intraperitoneal tumors in an immunocompetent syngeneic OC model. Radiolabeling with zirconium-89 generated [Zr]Zr-DFO-anti-TRA scFv-Fc with >98% radiochemical yield. Serial PET/CT imaging demonstrated progressive and sustained radiotracer accumulation at tumor sites through 96 hours, accompanied by declining liver-associated activity and low uptake in most normal tissues. Together, these findings identify TRA as a broadly expressed and accessible tumor-associated glycoepitope and establish TRA-targeted immunoPET as a promising strategy for noninvasive detection of OC. The selective and sustained tumor localization of this platform further provides a foundation for development of TRA-directed radiopharmaceutical therapy, supporting a potential theranostic approach for OC.

Background: Tumor necrosis factor- inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods: Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results: All three variants showed two- to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions: Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation.

The leukocyte-specific {beta}2-integrin receptor family exerts a wide range of functions: {beta}2-integrins are involved in leukocyte trafficking, where they mediate cell adhesion during inflammatory responses via binding to ICAM-1, ICAM-2, or JAM-C. Furthermore, they are essential for the recognition and phagocytosis of pathogens opsonized by complement. Accordingly, the {beta}2-integrin family is known to be involved in autoimmune and inflammatory diseases, such as systemic lupus erythematosus. Owing to their complex biology, involving multiple conformational transitions, different signaling pathways, and a broad spectrum of ligands, the development of {beta}2-integrin-targeted probes and therapeutics has remained challenging. We aimed to develop macrocyclic peptides, derived from phage display screening, which can be used to unravel ligand binding profiles of {beta}2-integrins with an emphasis on the I domain. The selection of suitable lead peptides, and the characterization of their interaction profiles with different I domains, was enabled by an established in-vitro assay platform. Various peptide sequences were enriched during several rounds of phage display against the I-domain of CR3, of which two peptides with particularly low micromolar binding affinity were further characterized. Both peptides showed direct binding to {beta}2-integrin I-domains and, in a competitive assay, dose-dependent inhibition of the I-domains interactions with their main ligands iC3b and ICAM-1, respectively. These ligand-interfering properties were confirmed in bead- and cell-based adhesion assays. The modulators developed here are expected to provide valuable insight into the (patho-)physiology of CR3 and the other members of the {beta}2-integrin family, as the two peptides were able to compete with different ligands. In the future, this may help to identify potential therapeutic approaches for autoimmune, inflammatory, and age-related diseases.

Many bacterial species naturally secrete membrane vesicles (MVs) that mediate the intercellular transport of biomolecules, including nucleic acids, proteins, and metabolites. Beyond their native physiological roles, MVs hold considerable potential for biomedical applications. Here, we demonstrate that MVs from several Bacillus species exhibit potent intrinsic adjuvant activity, efficaciously eliciting immune responses and facilitating antigen-specific antibody production in mice. Exploiting this adjuvanticity, we engineered a highly adaptable universal vaccine platform that uses B. subtilis MVs as self-adjuvanting carriers. This system employs a modular "plug-and-display" architecture that covalently anchors recombinant antigens to the MV surface through a multi-step bioconjugation cascade. After validation of this methodology using a model antigen, we adapted the platform to target Yersinia pestis, the causative agent of plague. We formulated a Y. pestis vaccine by labeling the MV surface with a modified capsule antigen fraction 1 (mCaf1). Intranasal administration of the mCaf1-MV vaccine effectively elicited both systemic and mucosal immunity. Crucially, this vaccine conferred highly efficacious protection against a lethal Y. pestis infection in a murine model. These findings demonstrate the exceptional protective efficacy of the B. subtilis MV platform and highlight its broad potential for the rapid development of mucosal vaccines against diverse emerging pathogens.

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.

Multiplexed imaging of biomolecular networks in living cells is limited by the small number of spectral separable fluorophores and the need to monitor dynamic processes in real time. Here, we present logicFRIES, a fluorogenic RNA (FR)-based platform that enables eight-plex live-cell imaging through programmable, logic-gated activation coupled with sequential fluorescence imaging. By integrating small-molecule-binding RNA aptamers into dye-activating fluorogenic RNAs, we engineered trigger-responsive FR reporters. This design implements an AND-gated mechanism in which fluorescence activation requires both a cognate trigger molecule and its corresponding fluorogenic dye, thereby expanding multiplexing capacity without adding new fluorophores. Using three membrane-permeable triggers, tetracycline, ASP2905, and guanine, we generated three distinct trigger-defined activation states for each engineered Broccoli and Pepper FR. Combined with orthogonal Corn/DFHO and DNB/TMR-DN reporter pairs, logicFRIES supports eight-plex imaging through sequential trigger/dye addition, imaging, and wash-based stripping cycles. We demonstrate robust, specific, and reversible fluorescence switching of these multiplexed FR reporters in living HEK293T and SKBR3 cells. Overall, logicFRIES extends live-cell imaging beyond conventional spectral limitations and provides a modular foundation for potentially developing multiplexed sensors targeting endogenous RNAs, proteins, and small molecules in complex cellular systems.

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

Background: Trophoblast cell surface antigen 2 (TROP2, encoded by TACSTD2) is a transmembrane glycoprotein overexpressed in multiple aggressive epithelial carcinomas. While antibody drug conjugates targeting TROP2 have achieved regulatory approvals, acquired payload resistance and systemic off-target toxicities limit sustained remissions. Radionuclide Drug Conjugates (RDCs) represent a potent alternative modality capable of delivering cytotoxic ionizing radiation directly to target cells. However, selecting the optimal therapeutic radioisotope between long-range beta emitters (177Lu) and short-range, high linear energy transfer (LET) alpha emitters (225Ac) under heterogeneous TROP2 spatial distributions remains an unaddressed clinical challenge. Methods: We developed an automated computational pathology and spatial microdosimetry pipeline to resolve microscopic TROP2 expression gradients and simulate absorbed radiation dose distributions from digitized whole-tissue immunohistochemistry (IHC) sections (N = 14). Optical density matrices were de-convoluted in Hematoxylin-Eosin-DAB (HED) color space to isolate the DAB chromogen. Continuous 2D spatial density distributions and topological surface profiles were reconstructed. Physical radiation energy deposition was modeled using radial dose point kernels for 177Lu (mean range ~670 m, LET 0.2 keV/m) and 225Ac (mean range ~65 m, LET 100 keV/m, 4 alpha particles per decay cascade). Therapeutic Index (TI, ratio of mean target to non-target absorbed dose), target coverage, and spatial specificity were quantified across all specimens. Results: Quantitative image deconvolution revealed that TROP2 expression across the cohort was characteristically focal and clustered, with a mean positive area fraction of 1.55 +/- 2.22% (range: 0.08% to 6.85%) and mean DAB signal intensity of 0.256 +/- 0.043. In all 14 evaluated specimens (100%), 225Ac-labeled RDCs demonstrated superior tumor-to-stroma dose localization compared to 177Lu-labeled RDCs. The cohort-wide mean Therapeutic Index was significantly higher for 225Ac (1.26 +/- 0.14) than for 177Lu (1.01 +/- 0.02, p < 0.0001, paired two-tailed t-test). Because the path length of 177Lu beta particles exceeded target cell nest dimensions by up to 30-fold, 177Lu suffered from severe off-target crossfire spillover into antigen-negative stroma. In contrast, 225Ac confined high-LET ionization tracks strictly within the micro-geographic boundaries of TROP2-expressing clusters. Conclusions: In tumors displaying focal or sparse TROP2 micro-architecture, Targeted Alpha Therapy with 225Ac-RDCs offers a superior biophysical profile over beta-emitting 177Lu-RDCs, maximizing cluster cell kill while sparing adjacent normal tissue stroma. This computational microdosimetry framework provides a practical tool to guide rational isotope pairing in RDC drug design.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.