Chemical Communications

Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR,sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, and most of them have suboptimal optical properties. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine (oxo-Ade BZT), as a new, remarkably bright and potent fluorescent universal nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M-1 x cm-1) originates from a high molar extinction coefficient (averaged{varepsilon} 368 37000 M-1 x cm-1), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 - 0.27). Melting temperature variations observed upon the incorporation of oxo-Ade BZT opposite native nucleobases in a duplex context did not exceed 10%. The basis of these universal hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-Ade BZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-Ade BZT-constituting heterobicycles decreases, and oxo-Ade BZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-Ade BZT a promising molecular tool for analytical, biophysical and biochemical studies.

In light of the "silent" AMR pandemic, new avenues to combat pathogenic bacteria are needed. In this work, we screened a large molecule library (n=35 684 unique compounds) with the aim of identifying molecules being able to bind and block translation of the prfA-thermosensor transcript in the bacterial pathogen Listeria monocytogenes. Using a thiazole-orange displacement approach, 468 ([~]1.3% of all molecules) showed the ability to reduce fluorescence. After dose response testing, 32 compounds remained promising and eight of them showed sufficient purity and availability to be further validated. Interestingly, four compounds, being structurally very similar, showed specificity for prfA at a varying degree. All four compounds carried 3 aromatic rings with one connecting amine between two of the rings and an amide linking an aliphatic amine side chain. The most selective compounds, M5, showed a Kd of [~]0.8 {micro}M for the prfA RNA at 35{degrees}C. However, none of the eight most efficient compounds were able to inhibit prfA translation in vitro, suggesting that the molecules are able to bind but not affect the stability of the overall structure. Through this work, we have been able to identify a set of molecules, able to bind the prfA thermosensor RNA selectively, but without affecting translation. These molecules could constitute an important scaffold for further drug development.

Antimicrobial photodynamic therapy (aPDT) is a promising alternative to antibiotics, yet the molecular factors determining bacterial susceptibility remain unclear. This study investigates the critical role of bacterial lipids, particularly cardiolipin (CL) in the aPDT response of Escherichia coli using methylene blue as a photosensitizer. Through genetic deletion ({Delta}clsABC) and chemical modification via mannitol supplementation, we demonstrate that reduced CL levels significantly enhance bacterial sensitivity, leading to an additional reduction in viability exceeding 3 log10. Quantitative lipidomics (MS,GC) confirmed substantial CL depletion and altered fatty acid saturation. Interestingly, while live CL-deficient cells were more vulnerable, biomimetic giant unilamellar vesicles (GUVs) with higher CL content showed greater susceptibility to photo-oxidation. These findings suggest that CL-rich microdomains in living bacteria act as functional scaffolds for stress-defense systems rather than mere targets for oxidative damage. Modulating membrane lipid composition thus represents a novel strategy to potentiate aPDT efficacy.

Developing bright and photostable red fluorescent proteins (RFPs) is one of the holy grails of the protein engineering community. Despite several attempts, finding such fluorescent proteins (FPs) has remained elusive. One bottleneck to engineering next generation RFPs is our lack of understanding of non-fluorescent or dark state properties in such constructs. Here, we develop a theoretical and experimental framework that describes how photobleaching decays in FPs relates to dark state conversion and ground state recovery. Our systematic photophysical investigation of mCherry and mCherry-d, an RFP with enhanced dark state behavior, showed the presence of photodestructive dark states in such FPs. Molecular dynamics simulations reveal enhanced fluctuation around the imidazolinone-end of the chromophore in mCherry-d, potentially facilitating conversion to non-fluorescent states. Collectively, this work quantifies dark state kinetics and gives insights into engineering dark states in RFPs to develop bright yet photostable molecular probes.

De novo melanin design seeks to extend natural melanin colors to new, stable colors (blue, purple, green) with sequence-to-color tunability. Natural melanin, polymerized from tyrosine (Y), is a robust pigment with heterogenous molecular weights. Control of melanin size (length) is challenging; thus, only specific colors (yellow to brown) exist in nature. In this work, we describe the design of blue melanin through the polymerization of Y-containing pentapeptides with two key properties: tight packing during peptide assembly and high solubility in aqueous environments. By motif scaffolding a pentapeptide-repeat protein (PRP) with RFdiffusion, we narrowed 160,000 possible combinations to a library of 905 Y-containing pentapeptides with tight packing features. Two of the most soluble designs successfully formed stable blue melanin with {lambda}max absorbing in 615-620 nm, contributed by homogeneous melanin length achieved around 60 Y units. Other designs also formed new colors (purple, green), along with more known colors (red, yellow, brown). We found that blue melanin exhibited thermal stability at an autoclave temperature of 121{degrees}C and photostability of weeks under 600 lux illumination. We also demonstrated the application of blue melanin as an electrophoretic ink. De novo color design from simple peptides could potentially transform how colorants are sourced and produced. Our approach with computational design should also inspire the development of new deep-learning tools to directly predict colors from amino acid sequences.

We developed a series of nitro reduction-reversible acylating reagents. Following optimization of the acylation conditions, these reagents were tested for deacylation with sodium dithionite in vitro. We applied this reversible acylation to modulate RNAzyme-mediated pre-tRNA maturation, demonstrating its ability to regulate RNA-RNA interactions. Furthermore, the in vitro reversible acylation of EGFP mRNA indicated effective control of its translational activity. To explore cellular applications, we validated NQO1-mediated deacylation in vitro and then induced hypoxia in HepG2 cells using cobalt chloride, thereby reactivating the function of acylated EGFP mRNA via endogenous NQO1. Overall, this study highlights the potential for developing nitro reduction-reversible acylation as a new strategy for RNA functional control and RNA-based drug modification.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) has been widely used to determine amyloid fibril structures at atomic resolution. Such studies typically rely on homogeneous fibril preparations that produce narrow linewidths and high spectral resolution, enabling reliable resonance assignment and structural analysis. However, many biologically relevant amyloid aggregates are structurally heterogeneous, resulting in spectral broadening and reduced sensitivity that hinder atomic-resolution characterization. Lipids are known to modulate amyloid aggregation pathways and promote the formation of toxic species that are often less homogeneous, further complicating NMR-based investigations. Here, we evaluate the feasibility of utilizing the benefits associated with high-field (1.1 GHz) SSNMR for studying ganglioside GD3-catalyzed A{beta}42 aggregates. Uniformly-13C,15N-labeled A{beta}42 was incubated with GD3 to generate lipid-associated aggregates and analyzed under MAS conditions. 13C cross-polarization magic-angle spinning (CPMAS) spectra and 2D 13C-13C chemical shift correlation experiments using CORD (COmbined R2nv-Driven) mixing were acquired and compared with data collected at 600 MHz. Despite the heterogeneous nature of the GM1-associated assemblies, the 1.1 GHz spectra exhibit enhanced sensitivity and improved spectral resolution. Better resolved resonances corresponding to selectively structured regions of A{beta}42 are observed, indicating the presence of an ordered core within the lipid-associated aggregates. These results demonstrate that ultrahigh-field SSNMR significantly improves the characterization of heterogeneous amyloid assemblies and provides a promising approach for atomic-level investigation of biologically relevant, lipid-modulated A{beta} aggregates.

Protein stability arises from a fine balance between stabilizing forces such as hydrophobic interactions, hydrogen bonding, and ionic interactions, and destabilizing contributions from solvent exposure and electrostatics. Although hydrophobic burial is the dominant driving force for folding, intra-chain hydrogen bonds and ionic interactions modulate stability in context-dependent ways, with effects that vary depending on their location and environment within the protein. Most studies of protein stability have focused on perturbations induced by pH, solvent composition, or mutations in protonated water, leaving the influence of solvent isotopes relatively underexplored. Notably, despite stronger hydrogen bonding in D2O, proteins exhibit diverse stability responses upon transfer from H2O to D2O, suggesting that differential hydration of nonpolar groups plays a key role. Here, the solvent isotope effect on protein stability is investigated using double-chain monellin (dcMN), a {beta}-sheet-rich, two-chain protein with well-characterized folding behavior. By combining conventional equilibrium unfolding measurements with hydrogen-deuterium exchange mass spectrometry (HDX-MS), the stability of wild-type and a less hydrophobic mutant (C42A) dcMN was compared in H2O and D2O, revealing greater stabilization of the wild-type protein in D2O and highlighting the importance of hydrophobic interactions in governing isotope-dependent stability.

Understanding lipid metabolism in peripheral glial cells is crucial for elucidating the molecular mechanisms underlying neurodegeneration, cancerogenesis and therapy resistance. Here, we introduce a spectrolipidomic sensing approach that integrates Raman, FT-IR, and AFM-IR spectroscopy to monitor nanoscale cholesterol remodeling in glial cells exposed to cannabidiol (CBD). Deuterated cholesterol (dChol) was employed as an intrinsic, spectroscopically active molecular probe, enabling selective tracking of cholesterol transformations through characteristic C-D vibrational signatures within the 2300-2000 cm-1 silent spectral region. Multimodal vibrational spectroscopy provided label-free, spatially resolved insight into lipid organization, redistribution, and metabolic reprogramming across micro- and nanoscales. The dChol probe enabled semi-quantitative evaluation of cholesterol uptake, esterification, and membrane integration, revealing that the sequence of CBD exposure, before or after probe addition, triggers distinct lipid metabolic pathways. Raman spectroscopy demonstrated superior sensitivity, with reliable detection of intracellular dChol at concentrations as low as 10 {micro}M, outperforming FT-IR imaging and confirming its suitability for cell lipid sensing. This analytical platform establishes deuterium-labeled lipids as powerful vibrational sensors for probing lipid metabolism and CBD-induced remodeling in situ. The presented spectrolipidomic framework paves the way for next-generation, spectroscopy-based biosensing systems capable of visualizing lipid dynamics, membrane restructuring, and drug- lipid interactions under pharmacological or environmental stress conditions. HighlightsO_LIDeuterated cholesterol (dChol) used as an intrinsic vibrational sensor C_LIO_LILower detection threshold of intracellular dChol for Raman than FT-IR C_LIO_LIAFM-IR reveals phases of lipid droplet formation in nanoscale C_LIO_LICBD alters cholesterol uptake, esterification, and lipid unsaturation profiles C_LI

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.

pH-sensitive fluorescent proteins (FPs) play a crucial role in investigating pH-related cellular processes, such as endocytosis and exocytosis. Existing pH-sensitive FPs generated from Aequorea victoria green fluorescent protein (GFP), such as superecliptic pHluorin (SEP) and Lime, have been widely employed to study these processes, but suffer from low photostability. Here, we report the development and characteristics of serapH, a genetically encodable pH biosensor with improved photostability compared to GFP analogues, which we generated using mStayGold as a scaffold. To aid in the development of serapH, we developed a method for screening pH-sensitive FP variants by directly evaluating both brightness and pH sensitivity in bacterial colonies on agar. This significantly increased the number of colonies that could be screened per round and reduced the time needed per round. The photostability of serapH should improve spatiotemporal resolution by increasing tolerance to higher excitation intensities and longer imaging durations, thereby expanding the range of applications of pH-sensitive FPs.

GPR52 is an orphan G protein-coupled receptor implicated in psychiatric and neurodegenerative disorders, but its endogenous ligand remains unidentified, limiting the exploration of its physiological functions and therapeutic potential. We pioneered a novel methodology for orphan GPCR ligand discovery utilizing the GPCR-activation-based (GRAB) strategy by developing GPR52-1.0, a genetically encoded fluorescent sensor. GPR52-1.0 exhibits excellent membrane trafficking and high sensitivity in HEK293T cells, cultured neurons, and acute mouse brain slices. Notably, it detects neuronal activity-dependent endogenous ligand release in the striatum, with responses abolished by a specific antagonist. This sensor provides a powerful tool for identifying GPR52s endogenous ligand(s) and enables real-time monitoring of its activation. Our work lays the foundation for uncovering GPR52s physiological roles and supports future efforts to develop GPR52-targeted therapeutics.

Polyethylene terephthalate (PET) is a commonly used plastic worldwide and reducing its prevalence is crucial to improving environmental pollution. PETase that degrades PET plastic have received a lot of attention recently. This paper evaluates the ester hydrolysis process under both acidic and basic conditions, and shows that the local environment of the protein active site takes advantage of both. High pH in the protein buffer creates a better nucleophile to attack the ester through a proton shuttle channel in the protein, while local hydrogen bonds to the carbonyl of the ester stabilizes the intermediate/transition state of the hydrolysis reaction. With the understanding at the atomic level, we propose two engineering directions that can potentially improve the reactivity of the PETase: 1) increase the alkaline stability of the protein in general; 2) perturb the local hydrogen bond network to increase the partial charge on the PET carbonyl to be hydrolyzed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/703441v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@151b69borg.highwire.dtl.DTLVardef@1abb95dorg.highwire.dtl.DTLVardef@116a225org.highwire.dtl.DTLVardef@ef2bb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bifunctional diazirine lipids are valuable tools for mapping protein-lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/700185v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@15d1641org.highwire.dtl.DTLVardef@6024e0org.highwire.dtl.DTLVardef@1503dcorg.highwire.dtl.DTLVardef@1b067bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

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

MagLOV2 is an engineered flavoprotein designed to have large changes in fluorescence intensity in response to weak magnetic fields. Here, we characterize the magnitude of these fluorescence changes, known as the "magnetic field effect," as a function of the strength of an externally applied magnetic field in E. coli colonies expressing MagLOV2. We observe that the magnetic field effect is positive at low magnetic fields, reaches a maximum positive value near 1 mT, and then decreases, reversing sign at approximately 2 mT. Furthermore, the effect starts to plateau above approximately 70 mT, with a decreased sensitivity of fluorescence changes to magnetic fields above this range. The non-monotonic behavior, as well as the diminished responsiveness to higher magnetic fields, are consistent with the changes in fluorescence being driven by electron spin-dependent chemical processes governed by the radical pair mechanism.

The pathological hallmark of Parkinson Disease (PD) is the formation of the protein alpha-synuclein (Asyn) into {beta}-sheet rich, self-templating fibrils in the brain. Since the first atomic structure of wild-type Asyn fibrils was determined nearly a decade ago, several other in vitro structures of hereditary mutant fibrils and structures derived from post-mortem diseased patient tissue have been determined by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and cryo-electron microscopy. These structures have not only expanded the library of structures available for computational modeling of drug binding and therapeutics development but have also given unprecedented insight into the disease specificity and structural polymorphism of Asyn fibrils. Here, we report the high-resolution SSNMR structure of the A30P hereditary mutant Asyn fibril, associated with early-onset PD. Our structural model is calculated using several thousand distance restraints derived from one sample, primarily sourced through 3D 13C-13C-13C correlation experiments. The structure adopts a Greek key topology yet does not include the P30 mutation site within the fibril core. We also introduce a comprehensive method for the rapid comparison of SSNMR spectra between Asyn polymorphs of known structure and validate the A30P fold. Lastly, we find that the structure is highly similar to many other experimental structures of both in vitro and ex vivo Asyn fibrils, including those with other hereditary point mutations, suggesting a conserved accessible fold.

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.

Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition of protein can impair therapeutic efficacy and contribute to cell death leading to local tissue damage. Although gangliosides--glycosphingolipids containing sialic acid residues--are known to modulate amyloid formation in neurodegenerative disorders, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin aggregation. Using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM), the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates have been characterized. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. CD and FTIR data analyses revealed that insulin in the presence of gangliosides formed non-fibrillar intermediates with distinct secondary structures: {beta}-sheet-rich globular clusters in presence of GD3 and -helical intermediates in GM3-treated samples. Cytotoxicity assays further demonstrated that ganglioside-induced aggregates are significantly less toxic to cells when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin oligomers retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/703542v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@5bf40eorg.highwire.dtl.DTLVardef@f400ddorg.highwire.dtl.DTLVardef@164dcd8org.highwire.dtl.DTLVardef@def4e7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGangliosides GD3 and GM3 accelerate insulin aggregation, forming non-fibrillar assemblies. C_LIO_LIGanglioside-bound insulin aggregates are less cytotoxic than fibrillar aggregates. C_LIO_LIDespite altered morphology, ganglioside-bound aggregates retain seeding ability. C_LI