Chemical Communications

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Tumour biomarkers such as CA125, CA15-3, CA19-9, AFP and CEA are routinely used in the oncology clinic to diagnose cancer, monitor response to therapy, and detect relapse. However, their quantification depends on immunoassay-based methods that are time-consuming, reagent-dependent, and poorly suited to resource-limited settings. Here, we present a machine learning-assisted ATR-FTIR spectroscopy approach for label-free tumour biomarker analysis to enable simple and rapid quantification at the bedside. Using principal component analysis (PCA), we first demonstrate that these five clinically relevant biomarkers are spectrally separable, with the protein-associated region (1200-1700 cm-1) providing the greatest discriminative information. We then develop partial least squares regression (PLSR) models to quantify CA125 in phosphate-buffered saline (R2 = 0.95) and in human serum across a clinically relevant concentration range, achieving reliable predictions at and above the clinical decision threshold of 35 U/mL. A semi-quantitative classification model further demonstrated robust identification of elevated CA125, with a macro-average sensitivity of 0.86 and specificity of 0.92. These results support ATR-FTIR spectroscopy as a rapid, reagent-free platform for cancer biomarker monitoring, with potential utility in resource-limited settings. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/714840v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1be9c03org.highwire.dtl.DTLVardef@f49e5eorg.highwire.dtl.DTLVardef@1c93e39org.highwire.dtl.DTLVardef@1141e6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

Flexible and biocompatible piezoelectric materials are crucial for next-generation wearable and bio-integrated electronics. In this work, we report a sustainable bio-composite film by incorporating lysozyme, a naturally abundant protein, into a polyvinyl alcohol matrix to achieve efficient electromechanical conversion. The composite exploits the intrinsic molecular dipoles of lysozyme, which are effectively stabilized and aligned within the polymer network. Under applied bending strain and vertical pressure, the film exhibits a pronounced piezoelectric response, as evidenced by time-dependent electrical measurements under forward and reverse bias conditions. The deformation of -helices and other helical structures within lysozyme induces dipole reorientation and charge separation, generating a measurable electrical output. In contrast, pure polyvinyl alcohol films show no detectable response, confirming the essential role of lysozyme in the observed piezoelectricity. Furthermore, the device enables real-time human motion sensing, highlighting its potential for flexible, eco-friendly, and biocompatible electronic applications.

Therapeutic peptides combine high target specificity with potent biological activity.1 However, treatment success is often limited by rapid clearance and the need for frequent injections.2, 3 This challenge is particularly acute for therapeutic peptides used in obesity, where clinical benefit must be balanced against dose-dependent adverse effects. In nature, these constraints are overcome by storing hormones as reversible fibrils,4 but pharmacokinetic control is essential for widespread adoption of bio-inspired self-assembled depots for therapeutic peptides. Here, we show that tuneable pharmacokinetics can be achieved and modelled by mapping the fundamental chemical parameters of reversibly self-assembly in vitro. We demonstrate this approach for the amylin analogue pramlintide. Amylin analogues are under development for the next generation of diabetes and obesity treatments, with improved mechanism of action e.g. preserving lean body mass.5-8 Pramlintide is an approved drug with a well-established safety profile, however, it has a comparable half-life to native amylin.8-12 In a pilot study, we achieve in vitro-in vivo correlation, increasing the half-life of pramlintide 20-82-fold in rats, while controlling burst release. These findings demonstrate that the optimisation of pharmacokinetics can be decoupled from peptide engineering, establishing a generalisable framework for generating long-acting peptide formulations by emulating native storage mechanisms.

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e56ffborg.highwire.dtl.DTLVardef@1ac8b5org.highwire.dtl.DTLVardef@6f23c1org.highwire.dtl.DTLVardef@945c54_HPS_FORMAT_FIGEXP M_FIG C_FIG

Direct solar-to-chemical conversion offers a compelling route to clean, dispatchable energy. Photosystem I (PSI), an evolutionarily optimized light-driven oxidoreductase central to oxygenic photosynthesis, can be repurposed for direct solar-fuel production by efficiently coupling its photochemistry to catalysts, thereby storing sunlight as chemical energy in the H-H bond of H2. One promising architecture integrates PSI with Pt nanoparticle (PtNP) catalysts to create photocatalytic PSI-PtNP biohybrids. Advancing these systems requires molecular-level insight into protein-nanoparticle interactions and the bio-nano electron transfer pathways that govern activity; however, progress has been constrained by limited structural data to guide rational design. Here, we present two molecular structures of active PSI-PtNP assemblies that (a) compare thermophilic and mesophilic PSI scaffolds and (b) probe how removal of the terminal [4Fe-4S] clusters and stromal subunits in PSI reshapes protein-nanoparticle interfaces and photocatalysis. Structural analyses and molecular dynamics simulations define the interface topology, electrostatics, and cofactor-to-nanoparticle distances, revealing key molecular features that control biohybrid formation and electron transfer efficiency. These data establish mechanistic links between scaffold composition, bio-nano interface geometry, and catalytic performance, yielding design principles for optimizing PSI-PtNP architectures. The resulting structure-function insights provide a blueprint for engineering PSI-based solar-fuels systems and, more broadly, inform the design of protein-nanomaterial interfaces for light-driven catalysis.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinsons disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinsons phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. Myo-inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. Scyllo-inositol is the second most abundant inositol isomer in the human brain and aberrant concentrations are associated with various diseases; however, its biological functions remain poorly understood. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism is reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol and responses of endogenous inositol isomers were measured in multiple cell lines by HILIC-MS/MS, showcasing the advantages of isotopic tracing. [13C6]scyllo-inositol proved to be a versatile isotopic tracer, when coupled with MS-based lipidomics and 2D NMR experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different cell lines. The results suggest a previously underappreciated role of scyllo-inositol in mammalian cells. The utilization of [13C6]scyllo-inositol will help to elucidate the role of scyllo-inositol metabolism in healthy and diseased states. SignificanceScyllo-inositol is a cyclic sugar alcohol found predominantly in the human brain. Changes in its concentration are associated with different diseases, and scyllo-inositol has been investigated as a potential drug against Alzheimers disease in clinical trials. However, its metabolic fate in mammalian cells is not well understood. We report here a synthetic strategy to obtain [13C6]scyllo-inositol and demonstrate, through isotopic tracing, its incorporation into phosphatidylinositols in different human-derived cell lines. This new stable isotopic tracer enables the investigation of the biological role of scyllo-inositol in mammals and beyond. HighlightsO_LIConcise synthesis of [13C6]scyllo-inositol C_LIO_LI[13C6]scyllo-inositol uptake and response of endogenous inositol isomers studied in multiple cell lines C_LIO_LIUse of [13C6]scyllo-inositol as an isotopic tracer in metabolomics and lipidomics experiments C_LIO_LIEvidence for scyllo-inositol incorporation into phosphatidylinositol in mammalian cells C_LI

Multidrug resistant (MDR) bacterial wound infections are an increasing clinical challenge and require alternatives to conventional antibiotics. Although antimicrobial proteins offer promise, their therapeutic use is limited by poor stability, proteolytic degradation, reduced activity under physiological conditions, and potential toxicity. This work reports pH-sensitive lipid nanocarriers composed of granulysin (GNLY) and oleic acid (OA) for antimicrobial delivery to infected tissues. At neutral pH, GNLY is retained within OA-based nanocarriers and protected from proteolytic degradation. At pH 5.0, such as in infected wounds, the carriers undergo structural reorganization and release GNLY, restoring antimicrobial activity. OAGNLY (32 {micro}g/mL) achieved >3-log reductions in Staphylococcus aureus and Escherichia coli within 1 hour, and up to 4-log reductions in Pseudomonas aeruginosa and Acinetobacter baumannii, at physiological salt concentrations where free GNLY was largely inactive. Minimum inhibitory concentrations were 16 {micro}g/mL for MRSA and 32 {micro}g/mL for colistin-resistant E. coli. Ultrastructural analysis using transmission electron microscopy revealed disruptions of bacterial membranes and intracellular structures following OAGNLY treatment. In a murine surgical wound infection model, topical application of OAGNLY for 4 hours reduced bacterial burden by >5 logs and significantly decreased inflammation, as confirmed by histological analysis. In parallel, OAGNLY demonstrated minimal cytotoxicity to mammalian cells at active concentrations. These findings identify OAGNLY nanocarriers as a promising platform for pH-responsive delivery of GNLY and highlight their potential application for treating MDR skin and soft tissue infections..

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.

Intrinsically disordered proteins (IDPs) represent major yet challenging therapeutic targets in neurodegenerative disease due to their conformational heterogeneity and aggregation-prone behavior. Tau protein is a prototypical IDP that forms pathological aggregates in Alzheimers disease and related tauopathies. Despite extensive clinical efforts, tau-directed monoclonal antibodies have demonstrated limited efficacy. Concurrently, single-domain antibodies (nanobodies) have been gaining importance because of their small size and membrane penetrating capabilities. New design paradigms are therefore required for nanobodies to enable precise targeting of disease-relevant conformations. Here, we describe a biophysical modelling and AI-guided nanobody discovery targeting the VQIVYK motif of tau, which constitutes the structural core of neurofibrillary tangles in Alzheimers Disease. Biophysical modelling-based target analysis identified low-energy conformational states of VQIVYK. These conformational insights were used to guide AI-driven nanobody design of CDR3 loops. Starting from a nanobody scaffold, we generated 145 candidate nanobodies through systematic backbone sampling and neural network-guided sequence design, followed by multi-dimensional computational prioritization. Two candidates demonstrated robust binding to synthetic full tau protein in ELISA binding assays, achieving binding indices of 148.9% and 140%, relative to reference controls. Notably, one candidate also exhibited strong reactivity in post-mortem Alzheimers disease human brain tissue, with a binding index of 236.1%, exceeding that of the positive control (222.9%). Structural analysis indicates that our nanobodies engineered CDR3 engages VQIVYK through optimized aromatic and hydrophobic interactions. Together, these findings establish a proof-of-concept for biophysics-guided, AI-guided nanobody engineering against IDPs and identifies them as a promising lead for tau-targeted single domain antibody development.

Cell-free expression systems (CFES) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation, as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity due to the additives. In this study, we present a CFES that contains a tardigrade-derived Cytosolic-Abundant Heat-Soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during low-cost room temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular for making CFES more accessible and portable. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/715078v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@8ecc2eorg.highwire.dtl.DTLVardef@ff0432org.highwire.dtl.DTLVardef@6c940eorg.highwire.dtl.DTLVardef@6c5390_HPS_FORMAT_FIGEXP M_FIG C_FIG

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.