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Development of the interplay between monomer sequence and supramolecular chemistry is critical if chemistry is to recapitulate the properties of proteins and nucleic acids in the synthetic world. We have created sequenced trimers of aromatic donor/acceptor units which participate in charge-transfer interactions, linked by phosphodiesters. Each sequence displays its own characteristic self-assembly, and moreover complementary sequences interact with each other to produce new nanostructures and emergent thermochromism. This finding paves the way towards new functional nanomaterials which make bio-analogous use of sequence to tune structure.

Ribonuclease HI (RNHI) non-specifically cleaves the RNA strand in RNA:DNA hybrid duplexes in a myriad of biological processes, including retroviral reverse transcription. Several RNHI homologs contain an extended domain, termed the handle region, that is critical to substrate binding. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have suggested a kinetic model in which the handle region can exist in open (substrate-binding competent) or closed (substrate-binding incompetent) states in homologs containing arginine or lysine at position 88 (using sequence numbering of E. coli RNHI), while the handle region populates a state intermediate between the open and closed conformers in homologs with asparagine at residue 88 [Stafford, K. A., et al., PLoS Comput. Biol.2013, 9, 1-10]. NMR parameters characterizing handle region dynamics are highly correlated with enzymatic activity for RNHI homologs with two-state (open/closed) handle regions [Martin, J. A., et al., Biochemistry2020, 59, 3201-3205]. The work presented herein shows that homologs with one-state (intermediate) handle regions display distinct structural features compared with their two-state counterparts. Comparisons of RNHI homologs and site-directed mutants with arginine at position 88 support a kinetic model for handle region dynamics that includes 12 unique transitions between eight conformations. Overall, these findings present an example of the structure-function relationships of enzymes and spotlight the use of NMR spectroscopy and MD simulations in uncovering fine details of conformational preferences.

As a biologically active macromolecule, deoxyribonucleic acid (DNA) has the advantages of sequence programmability and structure controllability and can accurately transmit sequence information to specific biological functions. Facing the complex internal microenvironment and heterogeneity in tumor treatment, the construction and applications of DNA-based nanomaterials have become a focus point of research. In particular, the hybridization of DNA molecules with other materials endows DNA-based nanomaterials with multiple functions such as targeting, stimulus responsiveness and regulations of biological activities, making DNA nanostructures great potential in the treatment of major human diseases. In this review, the construction and characteristics of DNA-based nanomaterials are introduced. Then, the functions and applications of DNA-based nanomaterials in the delivery of chemotherapy drugs and gene drugs, stimulus-responsive release and regulation of cell homeostasis are reviewed. Finally, the future development and challenges of DNA-based nanomaterials are prospected. We envision that DNA-based nanomaterials can enrich the nanomaterial system by rational design and synthesis and address the growing demands on biological and biomedical applications in the real world.

Motivation Deep learning has revolutionized protein tertiary structure prediction recently. The cutting-edge deep learning methods such as AlphaFold can predict high-accuracy tertiary structures for most individual protein chains. However, the accuracy of predicting quaternary structures of protein complexes consisting of multiple chains is still relatively low due to lack of advanced deep learning methods in the field. Because interchain residue–residue contacts can be used as distance restraints to guide quaternary structure modeling, here we develop a deep dilated convolutional residual network method (DRCon) to predict interchain residue–residue contacts in homodimers from residue–residue co-evolutionary signals derived from multiple sequence alignments of monomers, intrachain residue–residue contacts of monomers extracted from true/predicted tertiary structures or predicted by deep learning, and other sequence and structural features. Results Tested on three homodimer test datasets (Homo_std dataset, DeepHomo dataset and CASP-CAPRI dataset), the precision of DRCon for top L/5 interchain contact predictions (L: length of monomer in a homodimer) is 43.46%, 47.10% and 33.50% respectively at 6 Å contact threshold, which is substantially better than DeepHomo and DNCON2_inter and similar to Glinter. Moreover, our experiments demonstrate that using predicted tertiary structure or intrachain contacts of monomers in the unbound state as input, DRCon still performs well, even though its accuracy is lower than using true tertiary structures in the bound state are used as input. Finally, our case study shows that good interchain contact predictions can be used to build high-accuracy quaternary structure models of homodimers. Availability and implementation The source code of DRCon is available at https://github.com/jianlin-cheng/DRCon. The datasets are available at https://zenodo.org/record/5998532#.YgF70vXMKsB. Supplementary information Supplementary data are available at Bioinformatics online.

A bioinspired acid-triggered hemiacetalization/dehydration/[3 + 3]-type cycloaddition cascade process was disclosed, diastereoselectively furnishing furo[2,3-b]chromene skeleton under mild conditions. The viability of this approach was demonstrated by syntheses of a series of furo[2,3-b]chromene and pyrano[2,3-b]chromene derivatives. The successful total syntheses of two lignan-phloroglucinol hybrids, hyperaspidinols A and B, exemplified the synthetic utility of our biomimetic methodology.

Antibodies consisting of variable and constant regions, are a special type of proteins playing a vital role in immune system of the vertebrate. They have the remarkable ability to bind a large range of diverse antigens with extraordinary affinity and specificity. This malleability of binding makes antibodies an important class of biological drugs and biomarkers. In this article, we propose a method to identify which amino acid residues of an antibody directly interact with its associated antigen based on the features from sequence and structure. Our algorithm uses convolution neural networks (CNNs) linked with graph convolution networks (GCNs) to make use of information from both sequential and spatial neighbors to understand more about the local environment of target amino acid residue. Furthermore, we process the antigen partner of an antibody by employing an attention layer. Our method improves on the state-of-the-art methodology.

Driven by bioinspiration and appreciation of the structure of ericifolione, a biomimetic tautomerization/intermolecular inverse-electron-demand hetero Diels-Alder reaction cascade sequence promoted by sodium acetate to rapidly construct sterically hindered dihydropyran scaffolds was established, which allowed the first straightforward biomimetic total syntheses of ericifolione and its analogues with high simplicity. Moreover, this methodology set the stage for the preparation of relevant natural products or derivatives.

The selenium-substituted heteroarenes are biologically active compounds and useful building blocks. In this sequence, we have developed a metal-and oxidant-free, environmentally friendly protocol for the regioselective selenylation of 2H-indazole derivatives by electrochemical strategy. A number of selenylated 2H-indazoles with wide functional groups have been synthesized in moderate to good yields under mild and environment-friendly reaction conditions.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker-sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTLH1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a lower binding affinity for lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Hydrogels mimic the natural extracellular matrix in terms of their nanofibrous structure and large water content. However, the lack of a combination of properties including sufficient heterogeneity in the gel structure, intrinsic antimicrobial activity, and bioactivity limits the efficiency of hydrogels for tissue engineering applications. In this work, a hydrogel with a combination of these properties was fabricated by hybridizing silk fibroin with a low-molecular-weight peptide gelator. It was observed that silk fibroin and the peptide gelator assembled orthogonally in sequence. While the morphology of silk fibroin nanofibrils was not affected by the peptide gelator, silk fibroin promoted the formation of wider nanoribbons of the peptide gelator by modulating its nucleation and growth. Orthogonal assembly maintained the antimicrobial activity of the peptide gelator and the excellent biocompatibility of silk fibroin in the hybrid gel. The hybrid gel also demonstrated improved interactions with cells, an indicator of a higher bioactivity, possibly due to the heterogeneous double network structure.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.

Silica particles are widely used as versatile materials with applications in catalyst support, biomedical materials, drug delivery systems, coatings, and cosmetics. Silica particle formation by biological catalysts could be more suitable for biotechnological applications. Although silicatein (protein) and R5 (peptide) are well-known as silica-forming biomolecular agents, new biocatalysts which can mediate silica particle synthesis under mild conditions are still needed. Here, we introduced three new silica-forming peptides (SFPs) found by BLASTp search using silica binding motif (RRSSGGRR) as a query. The three peptides were found to mediate biosilica formation under microbial culture conditions at pH 6-7. Among them, the peptide from Winogradskyella arenosi (Wa-RSG) exhibited better performance than other SFPs in the generation of silica particles at pH 6, at which silica is not easily formed. We investigated the optimal amount and reaction time of Wa-RSG for biosilica particle formation at pH 6-7. In addition, we demonstrated that Wa-RSG adsorbed on Escherichiacoli cell surfaces could induce silica shell coating of E. coli at pH 6-7, and the biosilica-coated microbes showed stability against harsh UV treatment. These results will be useful for the preparation of bio-integrated silica materials such as biomolecule-containing silica particles or silica-coated industrial microbes.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

All-hydrocarbon stapling strategy has been widely applied for enhancing the proteolytic stability of peptides. However, two major technical hurdles to some extent limit the development of stapled peptides for therapeutic usage: rational selection of the stapling sites and the corresponding deletion of the native side chains. Previously we described the development of the olefin-terminated amino acids with the retention of native side chains and successfully applied them in the synthesis of hydrocarbon stapled peptides with single side-chain retention. Here, we explored the feasibility and effectiveness of hydrocarbon stapling strategy characterized as double side-chains retention. Modeled after a lengthy human immunodeficiency virus-1 (HIV-1) fusion inhibitor SC34EK, Leui, Seri+4 and Lysi, Leui+4 stapled peptides with the retention of double side-chains were effectively obtained. Our complementary study provided a convenient alternative to address where to install the staple in sequence for conventional all-hydrocarbon peptide stapling. Furthermore, this method not only conferred conformational reinforcement for SC34EK with high α-helicity and protease resistance, but also preserved the structural characteristic (key peripheral residues, charge and solubility) of the linear peptide to the maximum, which are crucial for anti-HIV-1 activity.

Unquestionably, polymers have influenced the world over the past 100 years. They are now more crucial than ever since the COVID-19 pandemic outbreak. The pandemic paved the way for certain polymers to be in the spotlight, namely sequence-defined polymers such as messenger ribonucleic acid (mRNA), which was the first type of vaccine to be authorized in the U.S. and Europe to protect against the SARS-CoV-2 virus. This rise of mRNA will probably influence scientific research concerning nucleic acids in general and RNA therapeutics in specific. In this Perspective, we highlight the recent trends in sequence-controlled and sequence-defined polymers. Then we discuss mRNA vaccines as an example to illustrate the need of ultimate sequence control to achieve complex functions such as specific activation of the immune system. We briefly present how mRNA vaccines are produced, the importance of modified nucleotides, the characteristic features, and the advantages and challenges associated with this class of vaccines. Finally, we discuss the chances and opportunities for polymer chemistry to provide solutions and contribute to the future progress of RNA-based therapeutics. We highlight two particular roles of polymers in this context. One represents conjugation of polymers to nucleic acids to form biohybrids. The other is concerned with advanced polymer-based carrier systems for nucleic acids. We believe that polymers can help to address present problems of RNA-based therapeutic technologies and impact the field beyond the COVID-19 pandemic.

Controlling monomer sequence and dispersity in synthetic macromolecules is a major goal in polymer science as both parameters determine materials’ properties and functions. However, synthetic approaches that can simultaneously control both sequence and dispersity remain experimentally unattainable. Here we report a simple, one pot and rapid synthesis of sequence-controlled multiblocks with on-demand control over dispersity while maintaining a high livingness, and good agreement between theoretical and experimental molecular weights and quantitative yields. Key to our approach is the regulation in the activity of the chain transfer agent during a controlled radical polymerization that enables the preparation of multiblocks with gradually ascending (Ɖ = 1.16 → 1.60), descending (Ɖ = 1.66 → 1.22), alternating low and high dispersity values (Ɖ = 1.17 → 1.61 → 1.24 → 1.70 → 1.26) or any combination thereof. We further demonstrate the potential of our methodology through the synthesis of highly ordered pentablock, octablock and decablock copolymers, which yield multiblocks with concurrent control over both sequence and dispersity. Synthetic approaches that can simultaneously control both polymer sequence and dispersity are difficult to achieve. Now, a switchable RAFT agent that regulates chain transfer activity during controlled radical polymerization has been shown to enable the one-pot synthesis of sequence-controlled multiblocks with on-demand control over dispersity while maintaining high livingness.

Transient amyloid intermediates are likely to be cytotoxic and play an essential role in amyloid-associated neurodegenerative diseases. Characterization of their structural and dynamic evolution is the key to elucidating the molecular mechanism of amyloid formation. Here, combining circular dichroism (CD), exciton couplet theory, and Fourier transform infrared spectroscopy with site-specific tryptophan (Trp) and its noncanonical derivative 5-cyano-tryptochan (Trp5CN), we developed a method to monitor strand-to-strand tertiary and sheet-to-sheet quaternary interactions in the aggregation cascades of an amyloidogenic fragment from protein SOD128-38 (with the sequence KVKVWGSIKGL). We found that the exciton couplet generated from the Bb band of Trp can be used as a probe for side chain interactions. Its sensitivity can be further improved by four times with the incorporation of Trp5CN. We further observed a red-shift of ∼2 cm-1 and a broadening of ∼2 cm-1 in the IR band generated from the CN stretch during the aggregation, which we attributed to the transition from a corkscrew-like structure to a cross-linked intermediate phase. We show here that the integration of optical methods with unique aromatic side chain-related probes is able to elucidate amyloid intermolecular interactions and even capture elusive transient intermediates on and off the amyloid assembling pathway.

Sequence-defined polymers prepared from vinyl monomers attract tremendous research interests in mimicking the precise primary sequence and remarkable biological functions of natural biopolymers. Se...

Hydrogels have attracted widespread attention for breaking the bottlenecks faced during facile drug delivery. To date, the preparation of jelly carriers for hydrophobic drugs remains challenging. In this study, by evaporating ethanol to drive the formation of hydrogen bonds, hydrophilic poly(vinyl alcohol) (PVA) and certain hydrophobic compounds [luteolin (LUT), quercetin (QUE), and myricetin (MYR)] were rapidly prepared into supramolecular hydrogel within 10 min. The gelation performance of these three hydrogels changed regularly with the changing sequence of LUT, QUE, and MYR. An investigation of the gelation pathway of these hybrid gels reveals that the formation of this type of gel follows a simple supramolecular self-assembly process, called "hydrophobe-hydrophile crosslinked gelation". Because the hydrogen bond between PVA and the drug is noncovalent and reversible, the hydrogel has good plasticity and self-healing properties, while the drugs can be controllably released by tuning the output stimuli. Using a rat sidewall-cecum abrasion adhesion model, the as-prepared hydrogel was highly efficient and safe in preventing postsurgical adhesion. This work provides a useful archetypical template for researchers interested in the efficient delivery and controllable release of hydrophobic drugs.

In immature oocytes, Balbiani bodies are conserved membraneless condensates implicated in oocyte polarization, the organization of mitochondria, and long-term organelle and RNA storage. In Xenopus laevis, Balbiani body assembly is mediated by the protein Velo1. Velo1 contains an N-terminal prion-like domain (PLD) that is essential for Balbiani body formation. PLDs have emerged as a class of intrinsically disordered regions that can undergo various different types of intracellular phase transitions and are often associated with dynamic, liquid-like condensates. Intriguingly, the Velo1 PLD forms solid-like assemblies. Here we sought to understand why Velo1 phase behavior appears to be biophysically distinct from that of other PLD-containing proteins. Through bioinformatic analysis and coarse-grained simulations, we predict that the clustering of aromatic residues and the amino acid composition of residues between aromatics can influence condensate material properties, organization, and the driving forces for assembly. To test our predictions, we redesigned the Velo1 PLD to test the impact of targeted sequence changes in vivo. We found that the Velo1 design with evenly spaced aromatic residues shows rapid internal dynamics, as probed by fluorescent recovery after photobleaching, even when recruited into Balbiani bodies. Our results suggest that Velo1 might have been selected in evolution for distinctly clustered aromatic residues to maintain the structure of Balbiani bodies in long-lived oocytes. In general, our work identifies several tunable parameters that can be used to augment the condensate material state, offering a road map for the design of synthetic condensates.

The determination of the block sequence of triblock copolymers is difficult without information on the synthetic conditions, such as starting materials and reagents used or analysis using tandem ma...

Oligoribonuclease (Orn), a member of the DEDDh superfamily, can hydrolyse 2–5 nt nanoRNAs to mononucleotides. It is involved in maintaining the intracellular levels of RNA, c-di-GMP signalling and transcription initiation in many bacterial species. Here, the crystal structure of Orn from Vibrio cholerae O1 El Tor (VcOrn) is reported at a resolution of 1.7 Å. VcOrn, which consists of nine α-helices and six β-strands, crystallizes with a single monomer in the asymmetric unit but forms a homodimer via crystallographic twofold symmetry. Electron density is observed in the active pocket that corresponds to an intersubunit N-terminal expression tag with sequence GPLGSHHH. The positively charged N-terminal tag binds in the negatively charged nucleotide-binding pocket with a buried surface area of ∼500 Å2. The N-terminal tag interacts with VcOrn via π–π stacking with two conserved residues involved in nucleotide binding, as well as via salt bridges and hydrogen bonds. The structure reported here reveals that the active pocket can accommodate polypeptides in addition to nucleotides, thus providing an important starting point for investigation into substrate modification and inhibitor design targeting VcOrn.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone‐mediated assembly of both tetrasomes and nucleosomes on the well‐defined Widom 601 positioning sequence using a co‐expression/reconstitution wheat germ cell‐free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell‐free platform, our method is expected to be useful for downstream applications in the field of chromatin research., In this study, we developed a novel histone chaperone‐mediated assembly method for tetrasomes and nucleosomes using a wheat germ cell‐free co‐expression system. Drosophila tetrasomes and nucleosomes were assembled on the Widom 601 sequence under near physiological conditions. Purified tetrasomes and nucleosomes were assessed by gel analyses and evaluated by atomic force microscopy.

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry-tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

The F420 deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although behaves as a nicotinamide cofactor due to its obligate hydride-transfer reactivity and similar low redox potential. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit great structural variability. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of the F420 -dependent oxidoreductases. Five different deazaflavoenzyme Classes (I-V) are described according to their structural folds as follows: Class I encompassing the TIM-barrel F420 -dependent enzymes; Class II including the Rossmann fold F420 -dependent enzymes; Class III comprising the β-roll F420 -dependent enzymes; Class IV which exclusively gathers the SH3 barrel F420 -dependent enzymes and Class V including the 3 layer ββα sandwich F420 -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes. This article is protected by copyright. All rights reserved.

The pH-low insertion peptide (pHLIP) and its analogs sense the microenvironmental pH variations in tumorous cells and serve as useful anticancer drug deliveries. The pHLIP binds peripherally to membranes and adopts random coil conformation at the physiological pH. The peptide switches from random coil to α-helical conformation and inserts unidirectionally into membrane bilayers when pH drops below a critical transition value that has been routinely determined by the Trp fluorescence spectroscopy. Recent high-resolution studies using solid-state NMR spectroscopy revealed the presence of thermodynamically stable intermediate states of membrane-associated pHLIP around the fluorescence-based transition pH-value. However, the molecular structural features and their mechanistic roles of these intermediate states in the pH-driven membrane insertion process of pHLIP remain largely unknown. This work utilizes solid-state NMR spectroscopy to explore 1) the mechanistic roles of key proline and arginine residues within the pHLIP sequence at intermediate pH-values, and 2) the changes in lipid dynamics at intermediate pH-values in multiple types of model bilayers with anionic phospholipid and/or cholesterol. Our results demonstrate several molecular structural and dynamics changes at around the transition pH-values, including the isomerization of proline-threonine backbone configuration, breaking of arginine-aspartic acid salt bridge and the formation of arginine-lipid interactions, and a universal decreasing of dynamics in lipid headgroups and alkyl chains. Overall, the outcomes provide important insights on the molecular interactions between pHLIP and membrane bilayers at intermediate pH-values and, therefore, prompt the understanding of pH-driven membrane insertion process of this anticancer drug-delivering peptide.

The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule., De novo designed peptide with β-hairpin structure assembles to form a β-barrelled nanopore that can detect not only polynucleotides but also polypeptide chains at a single-molecule level.

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics., Here the authors show that disordered polypeptide-RNA condensates exhibit rheological properties similar to that of a viscoelastic Maxwell fluid, and use simple polypeptide design rules to create microcondensates with tunable viscoelasticity.

By hybridizing fibers, natural fibers are widely employed to substitute synthetic fibers in all feasible applications. The present study focuses on the development and characterization of hybrid Ar...

DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.

RNA interactions are exceptionally strong and highly redundant. As such, nearly any two RNAs have the potential to interact with one another over relatively short stretches, especially at high RNA concentrations. This is especially true for pairs of RNAs that do not form strong self-structure. Such phenomena can drive liquid–liquid phase separation, either solely from RNA–RNA interactions in the presence of divalent or organic cations, or in concert with proteins. RNA interactions can drive multimerization of RNA strands via both base-pairing and tertiary interactions. In this article, we explore the tendency of RNA to form stable monomers, dimers, and higher order structures as a function of RNA length and sequence through a focus on the intrinsic thermodynamic, kinetic, and structural properties of RNA. The principles we discuss are independent of any specific type of biomolecular condensate, and thus widely applicable. We also speculate how external conditions experienced by living organisms can influence the formation of nonmembranous compartments, again focusing on the physical and structural properties of RNA. Plants, in particular, are subject to diverse abiotic stresses including extreme temperatures, drought, and salinity. These stresses and the cellular responses to them, including changes in the concentrations of small molecules such as polyamines, salts, and compatible solutes, have the potential to regulate condensate formation by melting or strengthening base-pairing. Reversible condensate formation, perhaps including regulation by circadian rhythms, could impact biological processes in plants, and other organisms.

Substituted polyhydroquinolines are ubiquitous skeletal cores found in drugs and bioactive natural products. As a new route to access this motif, we successfully developed a one-pot cyclization cascade with high chemocontrol and diastereoselectivity. The sequence generates two cycles, three carbon-carbon bonds, and an all-carbon quaternary center in a highly convergent process. Functionalized polyhydroquinolines and congeners can be accessed from commercially available amino acids. This versatile and robust strategy was applied to the synthesis of (±)-Δ7-mesembrenone.

Cell penetrating peptides (CPPs) hold immense potential for the transport of therapeutic agents to their active targets, due to their low cytotoxicity and high transduction efficiency. Adoption of CPPs as delivery systems, and development of novel peptides has been hampered by the variety of mechanisms and complexity of factors involved in cellular uptake. Quantitatively analyzing these systems is further hindered by the inability to compare data among reports due to varying experimental conditions. In this study we investigate the translocation of seventeen CPPs, representing cationic, amphipathic and hydrophobic physicochemical classes, through a DOPC membrane using molecular dynamics simulations. Free energy profiles for individual peptides inserting into the lipid bilayer were generated giving insight into both the approach and adsorption of different CPPs to cell membranes, and the feasibility of their direct translocation. The control of physical conditions and composition allows objective comparison of different peptides and analyze the effect of sequence on the adsorption and translocation energetics. Our results indicate that positively charged residues impart repulsion, while hydrophobic residues increase bilayer interaction. This study is the first step in fully understanding the processes and energetics involved in the passive translocation mechanisms of CPPs, which usually involve multiple peptides.

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic but non-disulfide bonded peptides known as the orbitides has been relatively ignored. What research has been conducted has focused on discovering bioactive orbitides, with less attention to structural features. A recently discovered subfamily of the orbitides known as the PawL-Derived Peptides (PLPs) are produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family’s structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive characterization of the PLP family. Nine PLPs were produced by solid phase peptide synthesis. Although orbitides can have antimicrobial and therapeutic effects, none of these nine PLPs showed antibacterial or antifungal activity. Their structural features were studied using solution NMR spectroscopy and seven were found to possess regions of backbone order. Ordered regions consist of β-turns, with some PLPs adopting two well-defined β-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse shapes.

In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria.

A novel supramolecular DNA hydrogel system was designed based on a directly synthesized chemically branched DNA. For the hydrogel formation, a self-dimer DNA with two sticky ends was designed as the linker to induce the gelation of B-Y. By programing the linker sequence, thermal and metal-ion responsiveness could be introduced into this hydrogel system. This supramolecular DNA hydrogel shows shear-thinning, designable responsiveness, and good biocompatibility, which will simplify the hydrogel composition and preparation process of the supramolecular DNA hydrogel and accelerate its biomedical applications.

The enantiomerically pure, bromobenzene-derived metabolite 5 has been transformed into enone 20 using a reaction sequence involving Suzuki-Miyaura cross-coupling and Eschenmoser-Claisen rearrangement processes. Treatment of compound 20 with lithium hydroxide results in an acetonide fragmentation reaction that delivers the 4,4-disubstituted cyclohexa-2,5-dienone 21, reductive de-oxygenation of which leads to congener 22. A closely related sequence of reactions can be used to convert the same homochiral starting material 5 into compound ent-22.

Herein we report unprecedented location‐dependent, size‐selective binding to designed lanthanide (Ln3+) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location‐dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N‐terminus can convert a selective site into a highly promiscuous one. An X‐ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location‐dependent metal selectivity within a coiled coil scaffold, as well as the first report of location‐dependent Ln3+ selectivity within a protein., Location‐dependent size selectivity can be programmed into designed metallo coiled coils, allowing for the generation of near‐identical selective and promiscuous metal‐ion sites. The first crystal structure of a lanthanide bound within a coiled coil reveals that the existence of an optimal radius stems from the structural constraints of the peptide scaffold. Therefore, a selective site can in principle be designed de novo for any given Ln3+ ion.

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.

Zwitterionic polymer brushes have broad applications in antifouling, biolubrication, and drug delivery. The charge distribution on polymers is critical to the structure and properties of surface-tethered zwitterionic polymer brushes. However, there is a lack of understanding of the relationship between the charge distribution and conformation in these systems, which is important for designing and predicting the functionality of controllable surfacetethered polymer brushes. Zwitterionic peptides with different sequences of charged amino acids are excellent model systems to elucidate such a charge−conformation relationship. By performing all-atom molecular dynamics (MD) simulations and developing a discrete-charge mean-field theory, we perform a systematic investigation on the effect of charge distribution on the conformations of zwitterionic peptide brushes. All-atom MD simulations reveal that the height of the peptide brush strongly depends on the distribution of the charges along the peptide chain. Contact map analysis reveals that the charge sequence also determines the preferred intrachain (loops and extended) and interchain (head-to-tail and parallel) structures. Through the theory developed by us, we show that the interchain electrostatic interactions are responsible for the contraction of peptide brushes with long charged blocks, while elasticity drives the contraction of peptide brushes with alternating-charged segments. This study provides a clear illustration of the factors influencing the sequence−conformation relationship of zwitterionic peptide brushes. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Accepted version M.L. and J.Y. thank the Singapore National Research Fellowship (NRF-NRFF11-2019-0004). B.Z. acknowledges the support of A*STAR under its AME YIRG Grant (Project No. A20E6c0100).

Herein we report an efficient and practical method to access chiral chromenones bearing one α-amino stereogenic center in the β position of the carbonyl group. The quinine-derived squaramide could efficiently promote Mannich/cycloketalization/dehydration tandem reactions between 1-(2-hydroxyaryl)-1,3-diketones and functionalized imines generated in situ, providing a wide range of chiral chromenones with propargylamine or α-amino ester moieties with good results (54 examples, up to 98% ee).

Viruses have evolved strategies to prevent apoptosis of infected cells at early stages of infection. The viral proteins (vBcl-2s) from specific viral genes adopt a helical fold that is structurally similar to that of mammalian antiapoptotic Bcl-2 proteins and exhibit little sequence similarity. Hence, vBcl-2 homologues are attractive targets to prevent viral infection. However, very few studies have focused on developing inhibitors for vBcl-2 homologues. In this study, we have considered two vBcl-2 homologues, A179L from African swine fever virus and BHRF1 from Epstein–Barr virus. We generated two sets of 8000 randomized BH3-like sequences from eight wild-type proapoptotic BH3 peptides. During this process, the four conserved hydrophobic residues and an Asp residue were retained at their respective positions, and all other positions were substituted randomly without any bias. We constructed 8000 structures each for A179L and BHRF1 in complex with BH3-like sequences. Histograms of interaction energies calculated between the peptide and the protein resulted in negatively skewed distributions. The BH3-like peptides with high helical propensities selected from the negative tail of the respective interaction energy distributions exhibited more favorable interactions with A179L and BHRF1, and they are rich in basic residues. Molecular dynamics studies and electrostatic potential maps further revealed that both acidic and basic residues favorably interact with A179L, while only basic residues have the most favorable interactions with BHRF1. As in mammalian homologues, the role of long-range interactions and nonhotspot residues has to be taken into account while designing specific BH3-mimetic inhibitors for vBcl-2 homologues.

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3’ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3’ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5’ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.

The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells. By conjugation with a cell-penetrating peptide and a lysosomal-sorting sequence, the resulting GlueTAC chimera triggered the internalization and degradation of programmed death-ligand 1 (PD-L1), which provides a new avenue to target and degrade cell-surface proteins.

The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences., Guzman-Luna et al. present a crosslinking analysis of the interaction of unstructured nascent chains with ribosomal proteins near the nascent polypeptide exit tunnel of the ribosome. They further analyze the dependence of these interactions on the peptide length, surface charge and ionic strength (including Mg+2 concentration), facilitating the understanding of co-translational protein folding and misfolding/aggregation.

Intracellular condensates formed through liquid–liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.

The formation of linear hydrocarbon chains by sequential coupling of C1 units on the metal surface is the central part of the Fischer-Tropsch (F-T) synthesis. Organometallic complexes have provided numerous models of relevant individual C-C coupling events but have failed to reproduce the complete chain lengthening sequence that transforms a linear Cn hydrocarbon chain into its Cn+1 homologue in an iterative fashion. In this work, we demonstrate stepwise growth of linear Cn hydrocarbon chains and their conversion to their Cn+1 homologues via consecutive addition of CH2 units on a molecular diruthenium carbide platform. The chain growth sequence is initiated by the formation of a μ-η1:η1-C═CH2 ligand from a C + CH2 coupling between the μ-carbido complex [(Cp*Ru)2(η-NPh)(μ-C)] (1; Cp* = η5-C5Me5) and Ph2SCH2. Then, the chain propagates via a general C═CHR + CH2 coupling and subsequent hydrogen-assisted isomerization of the resulting allene ligand μ-η1:η3-H2C═C═CHR to a higher vinylidene homologue μ-η1:η1-C═CH(CH2)R. By repeating this reaction sequence, up to C6 chains have been synthesized in a stepwise fashion. The key step of this chain homologation sequence is the selective hydrogenation of the μ-η1:η3-allene unit to the corresponding μ-alkylidene ligand. Isotope labeling and computational studies indicate that this transformation proceeds via the hydrogenation of the allene ligand to a terminal alkene form and its isomerization to the μ-alkylidene ligand facilitated by the coordinatively unsaturated diruthenium platform.

Although β-hairpins are widespread in proteins, there is still no universal tool to coax any small peptide to adopt a β-hairpin conformation, regardless of sequence. Here, we report that δ-linked γ(R)-methyl-ornithine ((δ)MeOrn) provides an improved β-turn template for inducing a β-hairpin conformation in peptides. We have developed a synthesis of protected (δ)MeOrn as a building block suitable for use in Fmoc-based solid-phase peptide synthesis. The synthesis begins with l-leucine and affords gram quantities of the N(α)-Boc-N(δ)-Fmoc-γ(R)-methyl-ornithine building block. X-ray crystallography confirms that the (δ)MeOrn turn unit adopts a folded structure in a macrocyclic β-hairpin peptide. CD and NMR spectroscopic experiments allow comparison of the (δ)MeOrn turn template to the δ-linked ornithine ((δ)Orn) turn template that our laboratory has previously introduced and also to the popular d-Pro-Gly turn template. Collectively, these studies demonstrate that the folding of the (δ)MeOrn turn template is substantially better than that of (δ)Orn and is comparable to d-Pro-Gly.