Journal of the American Chemical Society

Photo-induced protein labelling strategies have become essential tools in chemical biology, but most strategies require high energy wavelengths of light as input to drive reactivity. Recently, we reported a biocompatible method for engaging photo-induced electron transfer to drive protein labelling using biaryl pyridinium salts and, here, we report the design of a series of aromatic cation salts that trigger this process using longer wavelengths of light while maintaining a sterically minimal profile. We achieved this through the systematic study of structure-reactivity relationships of various donor-acceptor pyridinium salts possessing extended conjugation, and these studies revealed the need of a constrained trans-stilbene relationship between the probes donor and acceptor substituents in order to achieve protein labelling. Probes with chromene-based donor groups in particular showed either robust protein labelling, significant fluorescence quantum yields, or state-dependent photophysical properties; in turn enabling the same probes to be used for both photo-induced protein labelling and wash-free live-cell imaging. We also demonstrate that these enhanced probes possess robust reactivity in complex biological environments through green light-triggered intracellular labelling in live HeLa cells, resulting in the identification of 659 enriched proteins. This series of experiments not only demonstrates the ability of this latest generation of probes to engage in photo-induced labelling using lower energy light in complex proteomes, but also reveals new capabilities for photophysical state-dependent reactivity and measurements. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681063v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1be9a0eorg.highwire.dtl.DTLVardef@741221org.highwire.dtl.DTLVardef@525596org.highwire.dtl.DTLVardef@170ed11_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H2S-triggered 1,6-elimination of an -CF3-benzyl to release resorufin. In detecting intracellular H2S, 4-azido-(-CF3)-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF3 group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H2S in HeLa cells under stimulation conditions.

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

Nucleic acid triplexes are crucial structural motifs in gene regulation and biotechnology, yet the kinetic principles governing their formation remain poorly understood. While a stability hierarchy of RNA*DNA-DNA > DNA*DNA-DNA > RNA*RNA-RNA, with no DNA*RNA-RNA triplex forming, is known, the kinetic roles of terminal residues remain poorly understood. Here, we employ bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy to demonstrate that dangling ends from both the third strand (triplex-forming oligonucleotide, TFO) and the duplex dramatically enhance triplex stability. Kinetic analysis reveals this stabilization is primarily driven by a marked increase in the association rate (k{square}{square}). Crucially, creating a single-base-pair dangling end at either terminus of the duplex enhanced triplex stability more effectively than blunt ends. For example, DNA TFO dTFO5 binding to d(HP5+TA) was enhanced compared to dHP5, and similarly RNA TFO rTFO5 binding to RNA duplex r(HP5+UA) and DNA duplex d(HP5+TA) showed stronger affinity and faster association than to blunt-ended rHP5 and dHP5. Interestingly, removal of a terminal base pair from the blunt-end duplex, generating a TFO dangling end, also enhances binding affinity and association rate. This indicates that both duplex and TFO dangling ends provide critical nucleation platforms, while blunt-ended terminal triples are dynamic and contribute minimally to stability. Thus, our work establishes that optimal triplex formation requires strategic optimization of both TFO and duplex terminal structures through a fundamental kinetic principle (bimodal nucleation). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/686076v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1dc6950org.highwire.dtl.DTLVardef@1861387org.highwire.dtl.DTLVardef@16c24a5org.highwire.dtl.DTLVardef@da0bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDangling ends at both third strand and duplex termini enhance triplex stability by accelerating association C_LIO_LITriplex formation can be nucleated from either end with a bimodal association mechanism C_LIO_LITerminal blunt-end base triples are dynamic and contribute minimally to stability compared to tailored overhangs C_LI

Deubiquitinases (DUBs) are proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, which can lead to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many disease states, including cancer, neurodegeneration and inflammation, making them potentially attractive targets for therapeutic intervention. The >100 known DUB enzymes have been classified primarily by their conserved active sites, but we are still building our understanding of their substrate profiles, localization and regulation of DUB activity in diverse contexts. Ubiquitin-derived covalent activity-based probes (ABPs) are the premier tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which account for local DUB concentration, protein interactions, complexes, and organelle compartmentalization in intact cells or organisms. Here, through comprehensive warhead profiling we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12), a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cell systems. Through chemical proteomics and targeted assays we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs in live cells across a range of non-toxic concentrations, and in diverse cell lines and disease models, and we demonstrate its application to quantification of changes in intracellular DUB activity during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes. SYNOPSIS TOC Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/509970v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13285acorg.highwire.dtl.DTLVardef@1e602b1org.highwire.dtl.DTLVardef@1baeff6org.highwire.dtl.DTLVardef@1e00eb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

RNA-based genetic code is thought to be central to lifes emergence due to its dual ability for information transfer and catalysis. Nonetheless, the genetic code of early life was potentially not restricted to canonical genetic alphabets alone. The presence of an extensive repertoire of modified nucleobases in extant biology as signatures of the past, highlights the relevance of non-canonical alphabets, ably strengthened by experiments demonstrating their ready conversion into nucleosides and nucleotides. All these strongly support a pre-RNA World, wherein informational molecules are posited to have contained alternate genetic alphabets. Nevertheless, understanding pre-RNA molecules capacity to acquire emergent function has remained less prevalent. Further, the steps involved in their transition to a canonical RNA World has not been systematically studied in the origins of life framework. In this study, we report the synthesis of a prebiotically relevant genetic alphabet containing the non-canonical nucleobase, barbituric acid. We demonstrate for the first instance the enzymatic incorporation of this prebiotically plausible alphabet (BaTP) into an RNA, using proteinaceous T7 RNA polymerase. Pertinently, the incorporation of this genetic alphabet into a baby spinach aptamer did not affect its overall secondary structure, while also allowing it to retain its aptameric function. Furthermore, we demonstrate the faithful transfer of genetic information from pre-RNA-containing barbitudine nucleotides to DNA, using a high-fidelity RNA-dependent DNA polymerase. These findings allude to a putative pathway for the early molecular evolution of the genetic code of extant life.

Fatty acid (FA) modifications, such as enzymatic desaturation and elongation, have long been thought to involve sequential and highly specific enzyme-substrate interactions, which result in canonical products that are well-defined in their chain lengths, degree of unsaturation and double bond positions.1 These products act as a supply of building blocks for the synthesis of complex lipids supporting a symphony of lipid signals and membrane macrostructure. Recently, it was brought to light that differences in substrate availability due to enzyme inhibition can activate alternative pathways in a range of cancers, potentially altering the total species repertoire of FA metabolism.2,3 We have used isomer-resolved lipidomics to analyse human prostate tumours and cancer cell lines and reveal, for the first-time, the full extent of metabolic plasticity in cancer. Assigning the double bond position(s) in simple and complex lipids allows mapping of fatty acid desaturation and elongation via hitherto apocryphal metabolic pathways that generate FAs with unusual sites of unsaturation. Downstream utilisation of these FAs is demonstrated by their incorporation into complex structural lipids. The unsaturation profiles of different phospholipids reveal substantive structural variation between classes that will, necessarily, modulate lipid-centred biological processes in cancer cells including membrane fluidity3-5 and signal transduction.6-8

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click-chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

De novo protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses -helical barrel (HB) peptide assemblies, which comprise 5 or more -helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, HBs are analogous to molecular flasks made in supramolecular, polymer, and materials chemistry. Using Forster resonance energy transfer as a readout, we demonstrate that specific HBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an HB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the HB.

Targeted intracellular delivery of therapeutic peptides and proteins remains an important but unresolved goal in biotechnology. A promising approach is to engineer bacterial exotoxins that deliver their cytotoxic enzymes into cells and can be engineered to target cancer cells as is the case with immunotoxins. The well-studied diphtheria toxin translocation domain is ideally suited as a delivery platform as it has been shown to be capable of delivering a wide range of macromolecular cargo. Widespread deployment of DT-based therapeutics in humans, however, is complicated by the prevalence of pre-existing anti-DT antibodies from childhood vaccinations that reduce the exposure, efficacy and safety of this important class of protein drugs. Thus, there is a great need for delivery platforms with no pre-existing immunity in humans. Here, we describe the discovery and characterization of a distant diphtheria toxin homolog from the ancient reptile pathogen Austwickia chelonae that we have named Chelona Toxin (CT). We show that CT is comparable to DT structure and function in all respects except that it is not recognized by pre-existing anti-DT antibodies present in human sera. Moreover, we demonstrate that the CT translocase is superior to the DT translocase at delivering therapeutic protein cargo into target cells. These findings highlight CT as a potentially class-enabling new chassis for developing safer and more efficacious immunotoxins and intracellular protein delivery platforms for cancer therapy.

Phase transitions of proteins and nucleic acids (NA) leading to the formation of biomolecular condensates have been linked to various biological functions. Given the growing number of proteins/NA predicted to undergo liquid-liquid phase separation (LLPS), efficient tools to investigate this behavior are critical to advancing our understanding of biomolecular condensate function. The current standard used to study LLPS involves techniques that utilize exogenous fluorophore labels. The labeling process is often costly and time-consuming and comes with associated complexity that arises from unknown interactions from the bulky fluorescent tags. These aspects limit high throughput analysis of protein/NA phase separation based on external fluorophore labeling. Here, we report the discovery that intrinsic fluorescence, well into the visible spectrum, arises as an emergent property of biomolecular condensates. Leveraging this intrinsic fluorescence, we study condensate formation, directly measure their internal dynamics via Fluorescence Recovery after Photobleaching (FRAP), and examine the 3D morphology and transitions to various multiphase architectures. Through this approach, we find that a variety of G-quadruplex DNA readily form droplets with histone H1 and display dynamic exchange. In addition, we directly demonstrate that the 3D morphology, core-shell architecture, and sub-compartmentalization of condensate droplets are tunable via the charge ratio of components in solution and NA hybridization. Our method utilizes an inherent property of condensates, thus is broadly applicable to any phase-separated systems and can advance our understanding of biological phase transition.

Glyoxal (GO) is a highly reactive 1,2 dialdehyde implicated in the formation of a set of disease-associated post-translational modifications (PTMs) known as advanced glycation end products (AGEs). While GO has been widely reported to modify lysine to form the highly studied AGE carboxymethyllysine (CML), here we demonstrate that GO alone leads to highly chemoselective arginine glycation, yielding a stable glyoxal-derived hydroimidazolidine (GH-DH) product. This near-exclusively formed AGE has the same mass change as carboxymethylarginine (CMA), which implies that it may have been overlooked or misattributed in prior studies. In contrast, lysine modification by GO is highly dependent on the presence of hydride-based reducing agents, suggesting that prior reports may have artifactually generated CML through pervasive use of reductive amination protocols during sample preparation. These findings challenge the standing assumptions about the landscape of GO-derived glycation, emphasizing the importance of carefully considering the impact of experimental conditions in glycation studies. By redefining the complement of GO-derived AGEs, this study provides essential new information that is greatly needed for uncovering their biology, creating new tools for their study, and discovering therapies that ameliorate or mitigate their glycation-related damage.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile approach for small-molecule ligand discovery in native biological contexts. The covalent ligandability maps generated by ABPP that target cysteine have frequently leveraged the acrylamide as a reactive group due to its tempered electrophilicity and presence in many advanced tool compounds and therapeutics. More recently, alternative cysteine-directed reactive groups such as the butynamide have emerged as an additional source of covalent probes and drugs, but their global reactivity with the proteome remains largely unexplored. Here, we compare the ligandability maps of stereochemically defined acrylamide and butynamide compounds (stereoprobes) built from a common tryptoline core and find that the butynamides, despite exhibiting attenuated intrinsic and proteome-wide reactivity, preferentially engage a diverse set of proteins in human cancer cells. Among the butynamide-preferring proteins was C19orf54/ACTMAP, a cysteine protease required for the post-translational maturation of actin. We show that (1S, 3R)-tryptoline butynamides stereoselectively react with the catalytic nucleophile of ACTMAP, leading to accumulation of N-terminally unprocessed actin in cancer cells. Our findings support reactive group diversification as a strategy for expanding the ligandability of the human proteome and the butynamide, more specifically, as a differentiated cysteine-directed electrophile for chemical probe discovery.

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/154864v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e1a54forg.highwire.dtl.DTLVardef@12be680org.highwire.dtl.DTLVardef@129e027org.highwire.dtl.DTLVardef@3b2317_HPS_FORMAT_FIGEXP M_FIG C_FIG

Optically addressable spin systems, such as nitrogen-vacancy centers in diamond, have been widely studied for quantum sensing applications. In this work, we demonstrate that certain flavoproteins -- specifically cryptochrome and iLOV -- which generate spin correlated radical pairs upon optical excitation, also exhibit optically detected magnetic resonance (ODMR). Remarkably, the iLOV protein, commonly used in cellular imaging, displays ODMR contrast approaching 50%. We present initial applications including widefield magnetic field sensing and spatial modulation of photoluminescence using radiofrequency pulses and magnetic field gradients. Our results establish radical pairs in proteins as a novel platform for optically addressable spin systems, offering the key advantages of molecular designability and genetic encodability. Moreover, due to the spin-selective nature of radical pair chemistry, the results lay the groundwork for future radiofrequency-based manipulation of biological systems.

Diacylglycerols (DAGs) are central intermediates in lipid metabolism and signaling, yet their trafficking and persistence within lipid droplets (LDs) remain incompletely understood due to the lack of chemically stable, DAG-mimetic imaging tools. Here, we report the development of a family of solvatochromic fluorescent lipid analogs, termed DONDI, designed to probe DAG-associated dynamics at LDs. These probes are based on a 1,8-naphthalimide scaffold conjugated to modified aminoglycerol backbones bearing oleoyl chains to mimic native glycerolipids. Biophysical characterization and atomistic molecular dynamics simulations revealed probe-specific membrane insertion and hydrogen-bonding behaviors consistent with distinct lipid-mimetic properties. Live-cell imaging in NIH 3T3 fibroblasts demonstrated that DONDI probes were efficiently internalized and selectively accumulated within lipid droplets. Structure-function analysis identified DONDI-5 as the closest mimic of 1,2-diacylglycerol, displaying rapid uptake, strong LD enrichment, and prolonged intracellular retention without detectable relocalization to other cellular membranes. These properties enabled sustained visualization of LD-associated DAG pools over extended time scales. Collectively, this work establishes DONDI-5 as a chemically stable DAG-mimetic probe and provides direct experimental support that DAGs can be transported to and transiently stored within lipid droplets without prior conversion to triacylglycerols.

Bioorthogonal ligations encompass coupling chemistries that have considerable utility in living systems.1-3 Among the numerous bioorthogonal chemistries described to date, cycloaddition reactions between tetrazines and strained dienophiles are widely used in proteome, lipid, and glycan labeling due to their extremely rapid kinetics.4,5 In addition, a variety of functional groups can be released after the cycloaddition reaction,6,7 and drug delivery triggered by in vivo tetrazine ligation8 is in human phase I clinical trials.9 While applications of tetrazine ligations are growing in academia and industry, it has so far not been possible to control this chemistry to achieve the high degrees of spatial and temporal precision necessary for modifying mammalian cells with single-cell resolution. Here we demonstrate visible light-activated formation of tetrazines from photocaged dihydrotetrazines, which enables live-cell spatiotemporal control of rapid biorthogonal cycloaddition reactions between tetrazines and dienophiles such as trans-cyclooctenes (TCOs). Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic visible light to trigger tetrazine ligations on live mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that uncage prodrugs in response to tetrazine ligation, opening up new methods for photopharmacology and precision drug delivery using bioorthogonal chemistry.

Fluorine NMR is a powerful tool for probing biomolecular structure and dynamics, yet the performance of 19F probes is fundamentally constrained by rapid transverse relaxation driven by chemical shift anisotropy (CSA). Despite its central role, CSA has largely been treated as an immutable nuclear property rather than a chemically addressable design parameter. Here we demonstrate that the geometry of the CSA tensor - specifically its magnitude, symmetry, and orientation relative to the internuclear dipolar interaction - constitutes a decisive and engineerable determinant of relaxation behavior in coupled 19F-13C spin systems. Guided by electronic-structure calculations and Bloch-Redfield-Wangsness relaxation theory, we establish quantitative design rules that predict when CSA-dipolar interference can be exploited to suppress transverse relaxation. Implementation of these principles in a cysteine-reactive fluoropyrimidine scaffold yields a reporter that supports simultaneous 19F and 13C TROSY optimization, validated by solid-state MAS NMR and protein-based experiments. When incorporated into the 42 kDa maltose binding protein, the probe exhibits exceptionally slow 13C transverse relaxation (R2 {approx} 2-3 s-1) corresponding to linewidths of [~]2 Hz that persist even at apparent molecular weights exceeding 200 kDa. These results recast relaxation optimization as a chemically programmable problem and provide a general framework for the rational design of next-generation NMR probes tailored to large, dynamic, and heterogeneous biomolecular systems.

The value of microbial natural product pathways extends beyond the chemicals they produce, as the enzymes they encode can be harnessed as biocatalysts. Microbial type II polyketide synthases (PKSs) are particularly noteworthy, as these enzyme assemblies produce complex polyaromatic pharmacophores. Combinatorial biosynthesis with type II PKSs has been described as a promising route for accessing never-before-seen bioactive molecules, but this potential is stymied in part by the lack of functionally compatible non-cognate proteins across type II PKS systems. Acyl carrier proteins (ACPs) are central to this challenge, as they shuttle reactive intermediates and malonyl building blocks between the other type II PKS domains during biosynthesis. Activating ACPs to their holo state via the phosphopantetheinyl transferase (PPTase)-catalyzed installation of a coenzyme A (CoA)-derived phosphopantetheine (Ppant) arm is critical to effectively study and strategically engineer type II PKSs, but not all ACPs can be activated using conventional PPTases. Here, we report the discovery of a previously unexplored non-actinobacterial PPTase from Dictyobacter vulcani sp. nov. W12 (vulcPPT). We explored its compatibility with both native and non-native ACPs, observing that vulcPPT activated all ACPs tested in this study, including a non-cognate, non-actinobacterial ACP which cannot be activated by the prototypical broad substrate PPTases AcpS and Sfp. Strategic optimization of phosphopantetheinylation reaction conditions increased apo to holo conversion. In addition to identifying a promising new promiscuous PPTase, this work establishes a road map for further investigation of PPTase compatibility and increases access to functional synthase components for use in combinatorial biosynthesis.

The nonenzymatic replication of ribonucleic acid (RNA) oligonucleotides may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the laboratory with chemically activated nucleotides, but continued copying requires a source of chemical energy for in situ nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chemistry that activates nucleotides, but its application to nonenzymatic RNA copying remains a challenge. Here we report a novel pathway that enables the activation of RNA nucleotides in a manner that is compatible with template-directed nonenzymatic polymerization. We show that this pathway selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for nonenzymatic template-directed RNA copying. Our results will enable more realistic prebiotic chemical simulations of RNA copying based on continuous in situ nucleotide activation.