Angewandte Chemie

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Prostate cancer (PCa) is one of the principal contributors to health burden in the aging male population. PCa develops through dysregulation of androgen receptor (AR) signaling pathways. Despite improvements in diagnostic techniques and interventions, no pharmacological measures with long term efficacy have been established once PCa advances to castration resistant prostate cancer (CRPC). To circumvent this issue, tetra-aryl cyclobutanes (CBs) have been proposed as structurally distinct compounds with a mechanism of action differing from traditional androgen receptor signaling inhibitor (ARSIs). Here, we apply principles of crystal engineering and solid state synthesis to expand the class of CBs through strategic derivatization. The synthesis of the CB occurs quantitatively, producing no side products and eliminating the need for product purification. We demonstrate how head-to-tail stacking interactions of halo-pyrimidine rings can be exploited to stack and align unsymmetrical alkenes to undergo [2+2] photodimerization to generate the CB in the solid state. We examine the structure-function relationships of CBs in vitro by profiling AR mediated transcriptional activity, receptor translocation, and cell viability. Moreover, we explore and identify putative binding interactions within CB/AR complexes and establish an adaptive ligand-binding potential using molecular docking platforms. In total, our data suggests that CBs have unexploited therapeutic potential in CRPC and that green chemistry and crystal engineering principles offer a unique route to generating these drug candidates.

Deep learning-based structure prediction enables the design of peptide ligands without relying on naturally occurring scaffolds. However, most computationally generated peptides are not advanced beyond initial activity measurements, leaving the path to drug-like optimization and in vivo validation underexplored. Here we establish an end-to-end workflow for de novo peptide agonist discovery and maturation using the melanocortin-4 receptor (MC4R) as a model target. Using an AlphaFold2-based hallucination protocol implemented in ColabDesign, we generated more than 5,000 linear and head-to-tail cyclic candidate peptides directed towards the MC4R orthosteric pocket. Functional screening of a prioritized subset revealed measurable activity in 74% of linear peptides and 23% of cyclic peptides, from which we identified a cyclic agonist with an EC50 of 340 nM despite lacking the canonical melanocortin activation motif. We then performed systematic in vitro maturation by deep mutational scanning, half-life extender conjugation scanning, and a combinatorial optimization library, coupled with data-driven analysis to map sequence-activity relationships. These experiments identified an alternative activation motif centered on an APWR segment and yielded single-site variants with substantially improved potency. The most effective substitution, a proline at position 5, produced the E5P variant with an EC50 of 6.7 nM against the human melanocortin-4 receptor (hMC4R). Finally, central administration of E5P (10 nmol) reduced acute food intake in mice, providing in vivo proof of concept. Together, our results demonstrate a generalizable design-to-validation strategy for converting de novo peptide designs into optimized, pharmacologically active peptides, and expand the space of MC4R agonist chemotypes beyond endogenous melanocortins.

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

High-risk Human Papillomaviruses (HR-HPVs) are responsible for 5% of global cancers. While vaccines against HR-HPVs exist, there are no treatments available for individuals already infected. Cell-penetrating peptides (CPPs) have demonstrated antiviral properties against viruses by blocking viral entry and delivering antivirals into infected cells. Developing CPP-based therapies faces challenges including inefficient delivery of macromolecules and endosomal entrapment, which must be overcome for effective clinical application. This study identifies an HPV16 major capsid protein L1 derived cationic peptide as a potent CPP. Peptide uptake depended on both a cluster of cationic residues and the specific peptide sequence. Mechanistic studies showed peptide entry occurred via cell surface heparan sulfate-mediated, lipid-raft dependent endocytosis. The peptide efficiently delivered GFP into HaCaT keratinocytes, and associated with the Golgi apparatus, demonstrating endosomal escape. GFP fusion protein endocytosis relied on binding of the cationic peptide to cell surface heparan sulfates. Cell-penetrating ability was conserved among homologous regions of various HPV types. The peptide showed potent antiviral activity by inhibiting infection of HaCaT cells by several HR-HPV types collectively responsible for nearly all HPV-associated cancers. Excitingly, HPV18 L1-derived peptide from the homologous region exhibited potent antiviral activity against HPV16 by preventing viral internalization. Our findings characterize HPV-derived peptides as highly efficient CPPs with potential to deliver therapeutic agents into cells and assist in development of treatments for high-risk HPVs.

Acinetobacter baumannii is a top-priority, ESKAPE pathogen that poses a major challenge to human health. The pathogen is difficult to combat due to its extensive arsenal of antibiotic resistance and its protective polysaccharide capsule. In addition, A. baumannii isolates are highly heterogeneous, which complicates the development of rapid detection methods or novel targeted therapeutic approaches. Here, we discovered and characterized a new biotechnological tool, the nanobody H7 (NbH7), along with its conserved target, the surface-exposed Omp25 protein of A. baumannii, and elucidated their interaction at the molecular level. Moreover, we demonstrate that NbH7-functionalized magnetic beads enable selective and efficient capture of A. baumannii from bacterial mixtures, including non-pathogenic intestinal bacteria. This provides proof of concept for a new targeting system that remains effective across diverse A. baumannii clinical isolates and capsule types and holds potential for use in diagnostic cell enrichment and targeted therapies.

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.

Spatial organization and temporal regulation of membrane components are essential for achieving complex functions in artificial cells, such as cell division and signalling. DNA-based molecular tools provide a powerful means to control biomolecular interactions with high precision. Here, we investigate the phase behavior of cholesterol-modified, star-shaped DNA nanomotifs anchored to the lipid bilayers of giant unilamellar vesicles (GUVs), by using fluorescence confocal microscopy and cryo-electron microscopy. These motifs spontaneously anchor to the lipid bilayers via hydrophobic interactions and exhibit distinct spatial organization depending on their sticky end sequences. Motifs with complementary sticky end sequences interact and distribute uniformly, while orthogonal motifs with different sticky end sequences segregate into isolated gel-like domains with limited lateral mobility. Notably, the phase separation of motifs does not require lipid phase separation, indicating that DNA-driven organization can take place independently of lipid phase separation. The behavior of this system is governed by the interplay of three key parameters: (i) hydrophobic anchoring via cholesterol, (ii) electrostatic repulsion between negatively charged DNA nanomotifs, and (iii) sticky end interactions. The observed two-dimensional phase separation of orthogonal DNA nanomotifs at the GUV interface presents a novel strategy for controlling lateral membrane organization in GUV systems. This approach would offer flexibility in membrane composition and enables molecular positioning, thereby achieving a high degree of organization on the surface in artificial cell models.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prediction of G-quadruplexes (G4), noncanonical DNA secondary structures containing guanine tetrads, can have profound relevance across cancer, neurodegenerative and other genetic diseases, but require experimental validation by instrument intensive methods including CD spectroscopy, fluorescence, NMR, or crystallography. We report a novel, inexpensive, efficient and scalable 2-enzyme system for confirmation of G4 formation and copy number repeat count. First, QuadCleave, in which Mismatch Endonuclease I was used to repeatably, site-specifically and selectively cleave oligodeoxynucleotides containing exclusively G4-forming sequences. Second, HaeIII enzyme identified G4-forming sequences that can also generate GG/CC base pairs, and a sequences cleavage by both established it in C9ORF72, the most mutated gene in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These findings establish ubiquitously accessible experimental validation of G4 forming sequences and their copy number repeat assessment, with specificity to C9ORF72 oligodeoxynucleotides whose repeat expansion has critical clinical implications in a subpopulation of ALS and FTD patients.

Nanoscale lipid bilayer mimetics are powerful tools for research on lipid bilayer, membrane proteins or for drug delivery. Established nanoscale bilayer systems that are stabilized by short peptides or polymers produce a broad size distribution and are difficult to customize. Here we introduce a DNA nanotechnology-based lipid bilayer mimetic, in which we covalently conjugated established nanodisc-forming amphiphilic peptides to oligonucleotides. These peptide-DNA conjugates were then hybridized with a circular single-stranded scaffold to form stiff, circular PDC minicircles with 14 peptide modifications at the inner rim of the torus. Lipid reconstitution yielded defined nanodisc with a tightly controlled circumference and component stoichiometry. Molecular dynamics simulations further validated the structural stability and reveal an asymmetric migration of the DNA to one rim of the bilayer. To mimic membrane protein insertion, we co-reconstituted a transmembrane peptide coupled to a bulky quantum dot. In future applications, the size and peptide arrangement can easily be modified in these DNA-templated PDC nanodiscs.

Curcumin-functionalized gold nanoclusters are promising platforms for catalysis and drug delivery, yet the molecular determinants of their stability, morphology, and solvent response remain unclear. Here, microsecond all-atom molecular dynamics simulations are employed to investigate a 2 nm gold nanoparticle noncovalently coated with different curcumin forms, including neutral enol and trans-keto tautomers, the deprotonated enolate, and their mixtures in water-ethanol and water-methanol solvents. Layer-resolved analyses of radius of gyration, density profiles, and surface coverage reveal that neutral enol and trans forms generate compact assemblies with near-complete surface coverage, whereas enolate-rich systems adopt more expanded conformations with solvent-exposed molecules. Mixed systems preserve these intrinsic packing characteristics while improving overall coverage. Solvent substitution from ethanol to methanol reduces {pi}-{pi} stacking, strengthens Au-curcumin interactions, and increases surface coverage, yielding more compact nanostructures. Free energy and potential of mean force calculations indicate that deprotonated curcumin most effectively screens Au-Au interactions and stabilizes dispersed nanoparticles, while neutral tautomers provide moderate stabilization. Curcumin also enhances the loading of anticancer drug doxorubicin (DOX) onto Au nanoparticles, improving biocompatibility. Enolate(An)-containing systems produce extended structures with weaker membrane interactions, whereas neutral curcumin complexes form compact, positively charged assemblies that strongly bind to negatively charged cancer cell membranes. These findings clarify how tautomeric state and solvent environment cooperatively govern interfacial organization and colloidal stability, establish design guidelines for curcumin-based gold nanocarriers in catalysis, sensing, and drug delivery applications.

G-quadruplex (GQ) structures within the HIV-1 long terminal repeat (LTR) regulate viral transcription and represent promising antiviral targets; however, detailed mechanistic understanding of their ligand recognition at the molecular level remains limited and has largely been investigated under dilute conditions despite the crowded and compartmentalized nature of intracellular environment. Here, we investigate the interaction of the cationic porphyrin TMPyP4 with the HIV-1 LTR-III GQ under dilute conditions and inside protein-rich phase-separated condensates that mimic intracellular biocondensates. Steady-state and time-resolved fluorescence measurements reveal a dual binding behavior that is not discernible from absorption spectroscopy. A high-affinity guanine-rich binding mode leads to efficient fluorescence quenching through electron transfer from ground-state guanine to excited TMPyP4, whereas a weaker non-guanine binding mode gives rise to enhanced and long-lived emission. Nucleotide-specific control experiments validate the origin of these distinct binding environments. Molecular docking and molecular dynamics simulations further support preferential binding of TMPyP4 at the terminal G-quartet together with a secondary binding mode near the quadruplex-duplex junction. Importantly, both TMPyP4 and LTR-III GQ preferentially partition into the condensates, where the hybrid GQ structure, dual binding behavior, and associated excited-state signatures remain preserved despite the crowded and viscous environment. Although a slight reduction in binding affinity is observed inside the condensates, the overall binding mechanism remains largely preserved due to compensatory effects arising from the condensate microenvironment. Overall, this work demonstrates that ligand recognition of viral GQ remains preserved within protein condensates and establishes fluorescence spectroscopy as a sensitive approach for resolving hidden binding heterogeneity in GQ-ligand interactions.

Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

Several approaches are available to increase the efficiency of CRISPR/Cas9-based genome editing, including the co-inactivation of a gene that mediates the cytotoxic activity of a compound which can be used to enrich the population in edited cells. Here we show in multiple cell lines how inactivating HSD17B11, a non-essential Short-chain Dehydrogenase/Reductase, confers a strong resistance (29- to 130-fold resistance) in both human and mouse cells to a Phenyl diAlkynylCarbinol compound (PAC) without impacting cell viability and proliferation. We show how co-inactivating HSD71B11 along with selection with PAC is usable to quickly identify efficient guide(s) against a gene of interest and to readily isolate fully inactivated clones. Altogether, this work provides an experimental framework for the facile generation of knockouts using PAC for selecting successfully inactivated cells.

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

Liposomes are self-assembled lipid vesicles capable of encapsulating both hydrophilic and hydrophobic therapeutics, making them versatile platforms in drug delivery and biomedical technology. In this study, the limitations of the classical thin-film hydration method were critically evaluated, and a sustainable, systematically optimized strategy was established for generating defined liposomal lamellar phases. Hydration conditions were optimized, and 4 mL of buffer per 10 mg of lipid was determined to be optimal for effective rehydration and improved statistical reliability of vesicle measurements. A refined probe-sonication protocol (20% amplitude, 5 s ON/55 s OFF pulse) enabled controlled transformation of multivesicular vesicles into stable multilamellar and unilamellar vesicles at net ON-times of 90 s and 185 s, respectively, without overheating or contamination. In addition, a Python-based machine-learning tool was developed for vesicle size characterization. Collectively, these optimizations provided a reproducible and sustainable framework for preparing liposomes across different lamellar phases.