Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR–Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Å. Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 3 10 -helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

The 3C‐like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C‐terminal extra domain in addition to the chymotrypsin‐fold adopted by piconavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra‐domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained ≈ 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284–Thr285–Ile286 and another around Phe291) were discovered on which Ala‐mutations significantly increased the enzymatic activities. Based on this, a super‐active triple‐mutant STI/A with a 3.7‐fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild‐type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization‐critical residues plus Ser284–Thr285–Ile286 and Phe291 are clustered together to form a nano‐scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.

The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymatic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzymesubstrate interactions by use of the dynamic light scattering, circular dichroism, and NMR spectroscopy, we 1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and 2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; 1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and 2) although the chymotrypsin fold remained a monomer within a wide range of protein concentrations, the extra domain existed as a stable dimer even at a very low concentration. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymatic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the determination of the preferred solution conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.A disease with overall fatality rates of 14 -15%, characterized by high fever, malaise, rigor, headache, and nonproductive cough, suddenly appeared last year in southern China and then rapidly spread to other countries through Hong Kong (www. who.int/csr/sars/archive/2003_05_07a/en). The outbreak of this disease, now called severe acute respiratory syndrome (SARS), 1 was not only a worldwide health hazard but also resulted in great damages to both the regional and global economies. To combat this unprecedented challenge, governmental agencies and scientists all over the world worked together to identify its causative agent and to develop effective strategies to halt SARS. Consequently, a novel coronavirus was identified as the pathogenic agent of SARS and was, thus, called SARS coronavirus (1-2). On the other hand, neither an efficacious therapy nor a preventive treatment has been available to date despite tremendou...

Key Points• Global lncRNA discovery reveals novel erythroidspecific lncRNAs that are dynamically expressed and targeted by GATA1, TAL1, and KLF1.• Multiple types of lncRNAs promote red cell maturation by regulating neighboring loci, including DLEU2 and a novel Band 3 enhancer lncRNA.Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis. (Blood. 2014;123(4):570-581)

We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. 2 with a favorable surface-to-volume ratio; and (vi) the absence of a nucleus, mitochondria, and any DNA. Thus, any modification made to the DNA of RBC precursors is eliminated upon their enucleation and cannot lead to abnormal growth or tumorigenicity after their transfusion into a recipient.Engineered RBCs have been generated using encapsulation (2-4), by noncovalent attachment of foreign peptides, or through installation of proteins by fusion to a monoclonal antibody specific for an RBC surface protein (5, 6).However, modified RBCs have limitations if intended for application in vivo. Encapsulation allows the entrapment of sizable quantities of material but does so at the expense of disrupting plasma membrane integrity, with a concomitant reduction in circulatory half life of the modified RBCs. Osmosis-driven entrapment limits the chemical nature of materials that can be encapsulated successfully, the site of release is difficult to control, and encapsulated enzymes are functional only at the final destination, compromising reusability at other sites (5, 6). Targeting of cargo to RBCs by fusion to an RBC-specific antibody, (e.g., anti-glycophorin antibody), has limitations because this mode of attachment to the RBC is noncovalent and dissociates readily, thus reducing circulatory half life and mass of cargo available for delivery (5, 6). Other developments that exploit RBCs for targeted delivery include nanoparticles enveloped by an RBC-mimicking membrane and RBC-shaped polymers (1). The short in vivo survival rate of these RBC-inspired carriers (∼7 d maximum) may limit their therapeutic utility.There is a need to develop new methodology for engineering RBCs so that they can carry a wide variety of useful cargoes to specific locations in the body. We describe an approach that involves minimal modification of the RBCs, with preservation of plasma membrane integrity. The method involves sortase-mediated site-specific covalent attachment of payloads to specific RBC surface proteins.Bacterial sortases are transpeptidases capable of modifying suitably modified proteins in a covalent and site-specific manner (7,8). Sortase A from Staphylococcus aureus recognizes an LPXTG motif positioned close to the substrate's C terminus and cleaves between T and G to form a covalent acyl-enzyme intermediate. This intermediate is resolved by a nucleophilic N-terminal glycine residue on an appropriately designed probe (9) with concomitant formation of a peptide bond between substrate and probe. Conversely, a protein may be labeled at its N terminus by extending it with suitably exposed g...

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll ␤-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core ␤-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4-and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. The erythropoietin-producing hepatocellular (Eph) 3 carcinoma receptors constitute the largest family of receptor tyrosine kinases, with 16 individual receptors throughout the animal kingdom, which are activated by nine ephrins (1-6). Eph receptors and their ligands are both anchored onto the plasma membrane and are subdivided into two subclasses (A and B) based on their sequence conservation and binding preferences. Usually, EphA receptors (EphA1-A10) interact with glycosylphosphatidylinositol-anchored ephrin-A ligands (ephrin-A1-A6), whereas EphB receptors (EphB1-B6) interact with transmembrane ephrin-B ligands (ephrin-B1-B3) that have a short cytoplasmic portion carrying both Src homology domain 2 and PDZ domain-binding motifs (7,8).The Eph receptors have a modular structure, consisting of a unique N-terminal ephrin-binding domain followed by a cysteine-rich linker and two fibronectin type III repeats in the extracellular region. The intracellular region is composed of a conserved tyrosine kinase domain, a C-terminal sterile ␣-domain, and a PDZ-binding motif. The N-terminal 180-residue globular domain of the Eph receptors has been shown to be sufficient for high affinity ephrin binding (9 -11). EphA subclass receptors remarkably differ from EphB receptors because they lack a 4-residue insert in the H-I loop of the ligand-binding domain. Previously, the structures of the EphB2 and EphB4 ligand-binding domains have been determined in both the free state and in complex with ephrins or peptide antagonists (10,11,(12)(13)(14)(15). These studies have shown that the ligand-binding domains of EphB2 and EphB4 adopt the same jellyroll ␤-sandwich architecture composed of 11 antiparallel ␤-strands connected by loops of various lengths. In particular, the D-E and * This work was supported by National Medical Research Council of Singapore Grant R-154-000-382-213 (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must the...

Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a promising avenue to design specific inhibitors for 3CL proteases by disrupting their dynamic correlation network.