Chemical Communications

The methylation of cytosines at the 5th position (d5mC) is one of the most common epigenetic modifications, and alterations in methylation profile of cells are known to be involved in progression of many diseases including cancer. Increased stability of DNA accompanied by decreased flexibility upon methylation is thought to be a reason behind methylation profiles. The effects of d5mC on DNA stability and structure were investigated via systematic changes in the number and position of d5mCs in DDD. Our results revealed that d5mC substitutions changed DNA conformation and increased the stability only slightly. Next, the effect of DNA methylation on DNA-small molecule interactions was investigated using DDD and fully methylated analogue, DDD8. All the molecules examined (EtBr, Dox, Net and Hoe) had slightly higher affinity to DDD8 compared to DDD. Conversely, their effect, especially Doxs, on DDD structure was more pronounced. Further investigations via MD simulations revealed high selectivity of Dox towards a single intercalation site where the methoxy group of Dox interacts with the methyl group of d5mC and that of two precedent dT to create a highly stable hydrophobic cluster. Hydrophobic cluster formation was not observed upon Dox binding to DDD. Our results rationalize the increased stability of DDD8 over DDD, and open new routes for the design of drugs targeting epigenetic modifications. We suggest, the design of drugs that can engage in hydrophobic interactions with methyl groups in the major groove of a 5-dTdTd5mCdG-3 sequence might lead the way in specific targeting of hypermethylated regions in cancer cells.

Atomically precise gold nanoclusters (AuNCs) are ultra-small particles composed of ten to hundreds gold atoms and exhibit unique photophysical properties. Significant progress has been made in tuning and extending their luminescence in the near-infrared window through the design of AuNC assemblies. Herein, we report a straightforward method for synthesizing highly pure, programmable DNA tetrahedra functionalized with a controlled number of AuNCs (from one up to four AuNCs). Using ligand exchange chemistry, AuNCs bearing a single grafted ssDNA onto them were produced. These constructs then served as building blocks for synthesizing tetrahedra through DNA hybridization. Products obtained at each stage of the synthesis were thoroughly characterized using a range of complementary technics. Notably, mass spectrometry in native mode provided novel insights into the accurate composition and stoichiometry of these architectures. This study paves the way for the synthesis and the characterization of a variety of new three-dimensional, DNA-guided AuNC assemblies that may serve as powerful theranostics and biophotonic tools.

DNA-programmed assembly of plasmonic nanostructures provides a powerful route to couple molecular recognition with optical signal generation. Here, we report the sequence-specific assembly of DNA-functionalized gold nanorods using a sesame allergen-derived DNA biomarker as a molecular bridge. Target-induced assembly produces concentration-dependent assembly growth, plasmon coupling, and distinct assembly kinetics that are readily monitored by absorption spectroscopy, enabling label-free detection of the target DNA in the nanomolar concentration range. The assembled nanorods further produce strong surface-enhanced Raman scattering (SERS) signals arising from plasmonic coupling within the assemblies, extending detection to the picomolar regime without the use of Raman reporters. Quantitative analysis reveals that both the extent and rate of assembly formation are governed by target DNA concentration. These results establish a direct relationship between molecular recognition, assembly growth, plasmonic coupling, and spectroscopic response, highlighting DNA-programmed gold nanorod assembly as a versatile platform for investigating hybridization-driven plasmonic self-assembly and nucleic acid detection. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/732610v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@6a8f42org.highwire.dtl.DTLVardef@1e36b9corg.highwire.dtl.DTLVardef@1ade546org.highwire.dtl.DTLVardef@1a787bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Stereochemistry underlies structure-function relationships across biology and materials science, ranging from proteins to electronic and spintronic materials. In this work, we investigate the electron transport properties of different oligopeptide stereoisomers using experiments and computational modeling. Single-molecule electronic experiments show that stereochemical modifications in tyrosine-based peptides lead to significant variations in molecular conductance along the peptide backbone due to enhanced stacking interactions and electronic coupling of aromatic side chains. In addition, stereochemical variations in alanine-based peptides give rise to changes in conductivity due to secondary structure interactions arising from {beta}-turn conformations. All-atom molecular dynamics (MD) simulations and quantum mechanical calculations are used to understand the molecular origins of the effect of stereochemistry on the structural and electronic properties of peptides. Overall, this work shows that stereochemical modification of non-terminal amino acids effectively controls electron transport due to aromatic side chain interactions or secondary structure effects. These insights open new avenues for the molecular design of peptide-based electronic materials with enhanced function.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.

Triple-negative breast cancer (TNBC) remains a major therapeutic challenge due to the lack of effective molecular targets and the dose-limiting off-target toxicity of conventional chemotherapy. Here, we design and construct a mirror-image DNA (L-DNA) nanostructure functionalized with an epithelial cell adhesion molecule (EpCAM)-specific aptamer for targeted delivery of doxorubicin (DOX) to TNBC cells. The L-DNA nanostructure retains thermodynamic properties comparable to natural D-DNA while exhibiting substantially enhanced resistance to nuclease and serum-mediated degradation due to its mirror-image chirality. Thermal melting and serum stability assays confirmed superior structural stability of the L-DNA nanostructure compared to D-DNA counterparts. In vitro cytotoxicity studies demonstrated that the EpCAM-targeted L-DNA nanostructure has the potential to selectively inhibit the growth of EpCAM-positive TNBC cells while reducing cytotoxicity in normal cells. These findings demonstrate that combining aptamer targeting with mirror-image DNA nanotechnology provides a stable and selective nanoplatform for chemotherapeutic delivery, which can potentially improve the precision and therapeutic efficacy of treatment for aggressive breast cancers.

Actin turnover is a fundamental cellular process essential for cell dynamics, whose control is critical for both medicine and biotechnology. However, conventional small molecules modifying actin turnover scramble the structure of actin filaments and display high cellular toxicity. MICAL enzymes oxidizing methionine (Met) residues in actin can potentially address this challenge, but their large size and multiple required cofactors make MICALs manufacturing and utilization difficult. Here we show that redox-active chiral decavanadate nanoclusters with tartaric acid are capable of site-selective actin modulation, mimicking MICALs, while requiring no cofactors, displaying high biocompatibility and being membrane permeable. Decavanadate nanoclusters serve as atomically precise "nano-enzymes" oxidizing three Met residues in globular actin, including Met-176; the latter inhibits the opening of the backdoor segment and prevents depolymerization of actin filaments. The structure of actin filaments formed after nanocluster treatment revealed no structural disturbances as confirmed by cryo-electron microscopy. The biocompatibility and bioactivity of chiral decavanadate nanoclusters was demonstrated by modulation of actin in living NG108-15 cells. Taking advantage of atomically precise structure of the nanoclusters, we show that their docking into actin can be predicted computationally, indicating the possibility of programmable actin modulation using the tools of nanochemistry.

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44 nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two layered antiparallel chair type G quadruplex core with three edgewise loops and a terminal duplex, forming a G quadruplex duplex hybrid structure. Serotonin binds at the G quadruplex duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G quadruplex scaffold, which enables a pronounced binding triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

Tryptophan is a biologically important redox-active amino acid whose functions in proteins, including long-range electron transfer, protection against oxidative damage, and environmental sensing, are governed not only by its chemical identity but also by its precise structural organization. Inspired by this biological principle, we investigated whether controlling the organization of tryptophan within crystalline materials could provide a strategy for modulating its physicochemical properties and molecular accessibility. Using identical molecular components but distinct assembly pathways, tryptophan was organized either as a confined guest within a preformed Zn-imidazolate framework, yielding a star-shaped crystalline architecture, or as an integral coordination component during framework growth, producing a distinct layered Zn- tryptophan crystalline framework. Although assembled from the same building blocks, these two organization modes generated fundamentally different crystal structures, morphologies, and mechanisms of biomolecule incorporation. In both architectures, incorporation of tryptophan into the crystalline environment preserved its intrinsic fluorescence while producing robust fluorescence under multiple excitation wavelengths, highlighting the strong influence of molecular organization on its optical response. The structural modes also exhibited distinct encapsulation efficiencies and pH-dependent molecular accessibility, while secondary calcium-alginate fixation provided an additional level of control over guest retention without disrupting the underlying crystalline architecture. These results demonstrate that engineering the structural organization of tryptophan provides a versatile strategy for tuning the optical behavior, molecular accessibility, and functional integration of a biologically important redox-active amino acid in crystalline materials, establishing a foundation for future biomimetic redox architectures, responsive sensing platforms, and controlled molecular delivery.

Amino acids and peptides are promising building blocks for aqueous biomolecular CO2 capture systems, yet the coupled thermodynamics governing carbamate formation, proton transfer, carbonate speciation, and hydration remain difficult to resolve experimentally. Here, we establish isothermal titration calorimetry (ITC) as a quantitative platform for characterizing these coupled processes by integrating calorimetry with pH titrations, NMR spectroscopy, and a mechanistic thermodynamic model. Using L-lysine, L-arginine, and a series of Lys- and Arg-containing peptides, global fitting of ITC thermograms yielded thermodynamic parameters describing protonation and carbamate formation that accurately reproduced independent pH titrations and NMR-derived speciation. The analysis revealed that the characteristic biphasic calorimetric response originates from the coupled carbonate-amine equilibrium network and buffer collapse rather than carbamate saturation. Lys formed -, {varepsilon}-, and ,{varepsilon}-dicarbamates and exhibited more favorable apparent carbamate thermodynamics than Arg with the {varepsilon}-carbamate lying among the most favorable carbamate-forming amine sites reported for aqueous amines. Model-guided exploration of the fitted thermodynamic landscape further demonstrated that maximizing total CO2 retention, amine-mediated capture, and carbamate formation are distinct optimization problems governed by different combinations of pH, temperature, and CO2 loading. Extension to systematically spaced Lys-containing peptides showed that inter-amine separation alone does not control carbamate stability, highlighting the dominant role of the local thermodynamic environment in biomolecular CO2 capture. This work establishes ITC as a powerful experimental approach for extracting CO2-amine thermodynamics and provides a predictive framework for the rational design and optimization of amino acid-, peptide-, and protein-based carbon capture systems.

Halting the progression of neurodegenerative diseases remains one of the foremost challenges in medicinal chemistry due to the complex biology that drives disease progression. For example, a hallmark of synucleinopathies, such as Parkinsons disease, is the misfolding and aggregation of the protein -Synuclein (-Syn), driving the formation of toxic oligomers and fibrils that avoid natural intracellular clearance mechanisms, participate in unusual protein-protein interactions, and ultimately contribute to the death of dopaminergic neurons. The field of targeted protein degradation (TPD) has emerged as an innovative therapeutic route to selectively degrade proteins of interest that leverage natural intracellular protein degradation machinery. First generation TPD therapeutics have traditionally been designed as bifunctional, chimeric compounds in which a short covalent linker tethers a ligand designed to bind target proteins to a ligand that initiates an either proteosome- or lysosome-dependent protein degradation cascade. While initial studies have indicated the promise of these approaches, translation to the clinical setting has been challenging due to difficulties in achieving cellular internalization, long-term stability, and establishment of a generalizable strategy. To overcome these obstacles, this work has focused on adding modularity and dynamic capability to this classical model by leveraging a multivalent macromolecular approach to TPD. Specifically, peptide amphiphiles (PAs) were designed to self-assemble into high-aspect-ratio supramolecular nanofibers and present peptide epitopes on the surface of the fibers to target simultaneous binding of -Syn and recruitment of enzymes that facilitate entry into the lysosome-dependent chaperone-mediated autophagy protein degradation pathway. In vitro application of these bioactive PA nanofibers has demonstrated the ability to independently internalize in cells and reduce -Syn protein levels selectively and effectively. While further optimization of this model has the potential to be a viable therapeutic against -Syn aggregation, the modularity of these supramolecular nanofibers through facile monomer design and incorporation illustrates the potential of establishing a platform technology for targeting a diverse range of pathologic proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/739556v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d031adorg.highwire.dtl.DTLVardef@6d792dorg.highwire.dtl.DTLVardef@12e80c1org.highwire.dtl.DTLVardef@71a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

A platform method to associate native proteins and peptides onto a liposome membrane using site-specific N-terminal alkylation based on 1H-1,2,3-triazole-4-carbaldehyde (TA4C) is developed. The TA4C reagent reacts with the N-terminal -amino group of native proteins under mild aqueous conditions in a single step, without genetic engineering or protecting group strategies. Equipping TA4C with hexyl and nonyl chains provides a direct handle for tuning the association between the protein and membrane. N-terminal alkylation of green fluorescent protein (GFP) as a model proceeds in high yield (93% for the hexyl group and 70% for the nonyl group), and tethering the N-terminal alkyl group on GFP efficiently associates the protein with the liposomal membrane, as confirmed by confocal laser scanning microscopy and dynamic light scattering. We extended this strategy to an investigation of the GE11 peptide, a ligand for the epidermal growth factor receptor (EGFR). The liposome immobilized with GE11 peptide possessing an N-terminal alkyl group enables active targeting with EGFR-overexpressing A431 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/742023v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f4095org.highwire.dtl.DTLVardef@1c91509org.highwire.dtl.DTLVardef@841647org.highwire.dtl.DTLVardef@1d2909b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The formation of magnesium pyrophosphate (Mg2P2O7) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg2P2O7 serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg2P2O7 remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg2P2O7-driven co-aggregation mechanism involving glutathione-capped gold nanoclusters (GSH-AuNCs) that induces fluorescence enhancement, enabling real-time crystal formation monitoring. The mechanism was first investigated in simplified mixtures containing pyrophosphate (P2O74-) and magnesium (Mg2+) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg2P2O7 co-aggregation can be correlated with crystal formation/growth/solubilization and solution turbidity, while distinct kinetic patterns can be indicative of the crystal size at the end of the reaction. As a next level of complexity, we examined the effects of common components in an enzymatic amplification reaction, i.e., dithiothreitol (DTT), ammonium sulfate ((NH4)2SO4), deoxynucleotides (dNTPs) and Bst polymerase, on Mg2P2O7 formation through real-time GSH-AuNCs fluorescence variations. Guided by the above results, we studied and selected the experimental parameters for the design of an optimized qualitative (end-point) or quantitative (real-time) genetic test. Finally, the loop-mediated isothermal amplification (LAMP) was used as a platform to demonstrate the quantification of Influenza A RNA within the range of 102-108 copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of <25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg2P2O7 formation in other biotechnological processes, including in vitro transcription and Mg2P2O7-bioorganic composites synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/744482v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@96dd88org.highwire.dtl.DTLVardef@aa122dorg.highwire.dtl.DTLVardef@18f4abforg.highwire.dtl.DTLVardef@745f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

The function of nucleic acids is governed not only by their structure but also by their dynamics. At the molecular scale, transitions between functional structural states are superimposed on rapid thermal fluctuations, resulting in an intricate interplay that is challenging to resolve experimentally, particularly at the single-molecule level. Here, we introduce a novel approach for unraveling sub-microsecond dynamics in oligonucleotides, enabling direct observation of fluctuations in single DNA molecules. By immobilizing nucleic acids vertically on graphene and exploiting distance-dependent graphene energy transfer of fluorescent molecules attached to the DNA, we relate fluctuations in fluorescence intensity to biomolecular dynamics. We show that ionic strength modulates the fluctuations and that structural defects in DNA, such as nucleotide gaps or mismatches, alter the measured dynamics. The experimental findings are complemented by atomistic molecular dynamics simulations and kinetic Monte Carlo simulations, establishing a direct link between theoretical predictions of structure and dynamics and experimentally accessible fluctuation timescales. Overall, our findings advance the understanding of how thermal fluctuations affect oligonucleotides and are modulated by both external and internal stimuli.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the {beta}-sheet region of the B1 domain of protein G (GB1) as well as {beta}-strand interactions in amyloid fibrils of the Parkinsons disease protein -synuclein. In both contexts, we observe dramatic destabilization of the {beta}-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of -synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

Proteases are an important class of proteolytic enzymes having great importance in both basic science and industrial applications. While cells tightly regulate the spatio-temporal activity of different proteases for cellular homeostasis, mis-regulation often leads to adverse effects. In this context, the protease activity of papain and its activation by L-cysteine is poorly understood in the literature. Herein, we discover that the protease activity of papain can be effectively regulated via a spontaneous liquid-liquid phase separation (LLPS) pathway. We show that papain undergoes biomolecular condensation via spontaneous LLPS under macromolecular crowding through the involvement of intermolecular hydrophobic interactions. Secondary structure analyses revealed a compact conformation of phase-separated papain with increased -helix content. Although native free papain is found to be active towards synthetic and protein substrates, the proteolytic digestion produces heterogeneous peptide aggregates. In contrast, we found that papain droplets remain dormant toward protein digestion due to the disulfide linkage of the active cysteine residue (Cys-25) in its compact conformational state. More importantly, we show that the protease activity of phase-separated papain can be reactivated in the presence of L-cysteine to produce uniform soluble peptide fragments. Our findings indicate that although disulfide linkages are not necessary for the phase separation of papain, upon phase separation, intermolecular interactions between phase-separated papain result in the formation of disulfide linkages involving active Cys-25 residues. The present discovery has tremendous technological importance to boost the efficacy of meat tenderization in the food industry.

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.