Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Solid-phase peptide synthesis and solid-phase fragment coupling mediated by isonitriles

The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this chemistry to reiterative solid-phase peptide synthesis as well as solid-phase fragment coupling are described.Amide bond formations are arguably among the most important constructions in organic chemistry (1, 2). The centrality of the amide linkage, as found in polypeptides and proteins, in the maintenance of life hardly needs restatement. Numerous strategies, resulting in a vast array of protocols to synthesize biologically active polypeptides and proteins, have been demonstrated (3, 4). Central to reiterative polypeptide bond formations was the discovery and remarkable development of solid-phase peptide synthesis (SPPS) (5, 6). The extraordinary impact of SPPS in fostering enhanced access to homogeneous polypeptides is clear to everyone in the field.As we have described elsewhere, by classical, mechanistic reasoning, we were led to conjecture about some hitherto-unexplored possibilities relevant to the chemistry of isonitriles (7–14). It was anticipated that isonitriles might be able to mediate the acylation of amines, thus giving rise to amides (15). Early experiments focused on free carboxylic acids as the acylating agents. As our studies progressed, it was found that the combination of thioacids, amines, and isonitriles leads to the efficient formation of amide bonds under stoichiometric or near-stoichiometric conditions (7–13, 16, 17). Although there remain unresolved issues of detail and nuance, the governing mechanism for amide formation under these conditions involves reaction of the thioacid, 1, with an isonitrile, 2, to generate a thio-formimidate carboxylate mixed anhydride (thio-FCMA), 3, which is intercepted by the “acyl-accepting” amine to generate amide, 5, and thioformamide, 6 (Fig. 1). The efficiency of the amidation was further improved through the use of hydroxybenzotriazole (HOBt) (18), which could well give rise to HOBt ester 7, although this pathway has not been mechanistically proven.Open in a separate windowFig. 1.Isonitrile-mediated amidation; structure of OT.The potentialities of the isonitrile-mediated amidation method were foreshadowed via its application to the synthesis of cyclosporine (19). The power of the method was particularly well demonstrated in the context of our recent total synthesis of oxytocin (OT) (20), wherein isonitrile mediation was used in each of the peptide bond constructions, leading to the synthesis of the hormone in high yield and excellent purity. This nonapeptide is involved in a range of biological functions including parturition and lactation (21, 22). Signaling of OT to its receptor (OTR) is apparently an important factor in quality maintenance of various CNS functions (23). The ability to synthesize such modestly sized, but bio-impactful peptides in both native (wild-type) form, and as strategically modified variants, is one of the current missions of our laboratory, with the objective of possible applications to the very serious problem of autism (24–26).     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.Many viruses exhibit a high mutation rate when replicating their genomes, enabling quick adaptation to both changing cellular environments and therapeutics (1–5). Mammalian innate immune systems have developed a mechanism to exploit this high mutation rate against the virus; in a phenomenon termed “lethal mutagenesis,” (6–14) the immune system employs nucleic acid-modifying enzymes (e.g., APOBEC and ADAR) to increase the viral mutation rate sharply, stressing the functional gene product repertoire of the virus to the point that the viral population collapses (15–17). Several antiviral agents are proposed to work at least in part by a chemical version of lethal mutagenesis [e.g., ribavirin against hepatitis C virus (18–22), 5-hydroxy-2′-deoxycytidine against HIV (7), and T-705 against influenza viruses (23)]. When a sufficient number of these mutagenic nucleoside analogs is incorporated into viral genomes, the analogs increase the viral mutation rate above the error catastrophe limit, the rate above which no viable progeny are produced (6, 24–27). This work aimed to understand the molecular basis underlying the biological phenomenon of lethal mutagenesis induced by mutagenic nucleotides.The nucleoside analog 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212) (Fig. 1A) is specifically designed to induce lethal mutagenesis in HIV (28–30). KP1212, the only anti-HIV drug candidate in clinical trials to use this mechanism, has been shown to increase the mutation rate of HIV both in cell culture and in isolates from humans undergoing monotherapy (28, 29). The mutagenic properties of KP1212 in cell culture reveal that it is likely to base pair promiscuously with A and G, and that the progressive acquisition of mutations (primarily A-to-G and G-to-A transitions) precedes population collapse (Fig. 1B) (29). These data are supported by biochemical experiments performed using purified polymerases that establish the ability of KP1212 to pair with either A or G, both when the modified base enters DNA from the nucleotide pool and when it acts as a template base (30). Understanding the chemical and structural basis of mutagenesis of this drug candidate is critical for both its future clinical progress and the development of new therapeutic agents that work by the principle of lethal mutagenesis.Open in a separate windowFig. 1.Schematic presentation of KP1212''s mutagenic effect on viruses. (A) KP1212 exists as an array of different tautomeric forms, whereas cytosine almost exclusively exists as one form, the canonical keto-amino tautomer. (B) The deoxynucleotide analog of KP1212 is incorporated by viral polymerases, causing G-to-A and A-to-G mutations during viral replication. KP1212 is a poor substrate for human polymerases, which provides selectivity in its action against the virus. The progressive acquisition of mutations in the viral genome leads to viral population collapse.Tautomerism of KP1212 leading to viral mutagenesis has been proposed by us and others (29, 30) to be the basis for the clinical activity of this drug candidate. There are, however, no direct data to support that view. Tautomerism as the basis of mutagenesis of natural bases has long been proposed (31–35), and substantiated in part by experimental evidence of minor tautomeric forms of both canonical bases (36–38) and certain base analogs (e.g., 5-hydroxy-2′-deoxycytidine) (39). In a search for a chemical rationale to explain the ambiguous pairing of KP1212 during replication, the present study revealed that the compound readily adopts multiple tautomeric forms, some of which were unexpected. Previously, spectroscopic methods (e.g., UV, Raman, NMR) have been used to study tautomerism of nucleobases (37, 39, 40). In the current work, we also used a battery of spectroscopic tools (1D, 2D, and variable temperature NMR; FTIR; and 2D IR) (41) to quantify and structurally characterize the array of tautomers exhibited by KP1212. Tautomer interconversion equilibria deconvoluted from NMR spectra provided data on the relative levels of tautomers in solution. In parallel with the spectroscopic studies, the qualitative and quantitative features of KP1212 mutagenesis were directly determined by inserting the KP1212 base into a single-stranded viral vector and measuring the intrinsic mutagenic properties of the base, both in vitro and in vivo. Finally, a model is proposed that correlates the mutagenic and clinical properties of KP1212 with its ability to exist as multiple tautomers.     

Human genome–guided identification of memory-modulating drugs

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory—a trait central to posttraumatic stress disorder—and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand–receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand–receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.Recent advances in human genetics have led to an unprecedented rate of discovery of genes related to complex human disease, including neuropsychiatric disorders (1–3). The human genome–based gain of knowledge is certainly expected to have a large impact on drug discovery in complex human disease (4–6). It is, however, still not clear to what extent this knowledge can be used as a starting point for the identification of druggable molecular pathways of complex traits (7), including mental disorders (8).Genomewide association studies (GWASs) using single-marker statistics have been very successful in identifying trait-associated single-gene loci (9). It is, however, widely accepted that single marker–based analyses have limited power to identify the genetic basis of a given trait, as for example, many loci will fail to reach stringent genomewide significance threshold, despite the fact that they may be genuinely associated with the trait. Triggered by statistical approaches for the analysis of gene expression, gene set–based analytical methods have recently become available. These methods aim at identifying biologically meaningful sets of genes associated with a certain trait, rather than focusing on a single GWAS gene locus (10). By taking into account prior biological knowledge, gene set–based approaches examine whether test statistics for a group of related genes have consistent deviation from chance (10). As shown recently in studies on autism (11), bipolar disorder (12, 13), attention deficit hyperactivity disorder (ADHD) (14), and schizophrenia (15), such approaches can convincingly identify convergent molecular pathways relevant to neuropsychiatry. Importantly, the identification of groups of functionally related genes is likely to facilitate drug discovery, because the most significant single loci from a GWAS might not be the best candidates for therapeutic intervention (7, 10).In the present study, we focused on emotionally aversive memory—a trait central to anxiety disorders such as posttraumatic stress disorder (PTSD) (16–23). Strong memory for emotionally arousing events can be seen as a primarily adaptive phenomenon, which helps us to remember vital information (e.g., dangerous situations). In case of an extremely aversive event, however, this mechanism can also lead to intrusive and persistent traumatic memories, thereby contributing to the development and symptoms of PTSD (18–22). Symptoms related to aversive memory include intrusive daytime recollections, traumatic nightmares, and flashbacks in which components of the event are relived. Aversive memory is a genetically complex trait as shown both in healthy subjects and in traumatized individuals (17, 23). Furthermore, we recently reported evidence suggesting a genetic link between the predisposition to build strong aversive memories and the risk for PTSD (16).Based on these observations, we developed a process (Fig. 1) aimed at identifying gene sets related to human aversive memory, followed by a pharmacological intervention study as proof-of-concept for the genome-guided identification of memory-modulating drugs.Open in a separate windowFig. 1.Drug discovery process.     

Aptamer photoregulation in vivo

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.Aptamers are single-stranded nucleic acids that have emerged as a promising class of therapeutics owing to their relative ease of synthesis and high affinity and selectivity toward a range of targets including small molecules, proteins, viral particles, and living cells (1–6). Aptamers can fold into well-defined conformations and are more resistant to enzymatic degradation than other oligonucleotides (7–9). Aptamers have been suggested for imaging applications because their relatively small size and molecular mass (∼10 kDa) allow fast tissue penetration and clearance from blood (10, 11). The same characteristics make aptamers promising for effective delivery of diagnostic and therapeutic agents to tissues or organs. However, nonspecific accumulation of aptamers in normal tissues is undesirable (12–15) because it diminishes the proportion of aptamer that targets the desired tissue. This can adversely affect the therapeutic index of the aptamer; this may be particularly true if the aptamer is conjugated to a drug or drug delivery device. Moreover, aptamers themselves can have nonspecific toxic effects (16, 17). Ideally aptamers would achieve a high concentration in a pathological tissue of interest while maintaining low levels elsewhere. The activity of aptamers can be modulated in vivo by binding to polymers or complementary oligonucleotide sequences (18–20), but spatiotemporal regulation of aptamer activity in vivo has not been achieved, whereby activity would be enhanced in target tissues and not others. Here, we report a strategy to provide light-triggered control of aptamer function and distribution in vivo.Light is an excellent means of providing external spatiotemporal control of biological systems (21–26). Many strategies have been developed to incorporate photosensitive groups in nucleotides that can control cellular function or affect biological pathways or gene expression by light (24–29). Of particular interest, such approaches can be used to provide spatiotemporal control of gene activation (24). Here we hypothesized that light triggering can be used to achieve spatiotemporal control of binding of an aptamer injected systemically to its target tissue in vivo, which would have implications for control of delivery of therapeutic aptamers and/or conjugated drugs or drug delivery systems. We designed a photo-triggerable system whereby the aptamer of interest is inactivated by hybridization to a photo-labile complementary oligonucleotide. Upon irradiation, the complementary sequence breaks down, releasing the functional aptamer (Fig. 1). The aptamer of interest is the single-stranded DNA 26-mer aptamer AS1411 (A₁₄₁₁; sequence: 5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) that binds with high affinity and selectivity to nucleolin (30–32), which is overexpressed on the cell membrane of several types of cancer cells, including the 4T1 breast cancer cells used here (33–35). A₁₄₁₁ has been used for cancer targeting in vitro and in vivo (35, 36). A complementary photo-triggerable inhibitory oligonucleotide (OliP) was designed [sequence: 5′-CCA CCA//CCA CCA//CAA CCA C-3′, where // indicates photo-labile 1-(2-nitrophenyl)ethyl bonds (37); Scheme S1].Open in a separate windowFig. 1.The AS1411 aptamer (A₁₄₁₁, red DNA strand) is hybridized to a complementary oligonucleotide (OliP, green DNA strand) containing photo-cleavable bonds (black dots). The hybridized complex cannot bind cells. The A₁₄₁₁ can be released by light-triggered breakage of the OliP, allowing binding to cell surfaces.     

Shape control and compartmentalization in active colloidal cells

Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core–shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble–crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non–momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier–Stokes equation.Active matter describes particulate systems with the characteristic that each “particle” (agent) converts energy into motion (1, 2). Active matter covers a range of length scales that include molecular motors in the cytoskeleton (3–5), swimming bacteria (6–8), driven colloids (9, 10), flocks of birds and fish (11–14), and people and vehicles in motion (15). Over the last decade, studies of active matter have demonstrated behavior not seen in equilibrium systems, including giant number fluctuations (16, 17), emergent attraction and superdiffusion (18–20), clustering (21, 22), swarming (23–27), and self-assembled motifs (28, 29). These systems provide interesting theoretical and engineering challenges as well as opportunities to explore and target novel behaviors that proceed outside of thermodynamic equilibrium.Of particular interest are systems found in nature or inspired by natural phenomena. Biological systems usually operate in confined regions of space––think of intracellular space, interfaces and membranes, and the crowding of cells near surfaces. The role of hydrodynamics in confinement has been studied for biological swimmers, such as bacteria and sperm, showing accumulation at the walls (30–32) and upstream swimming along surfaces (33) or in a spiral vortex (34–36). Attraction to walls has also been reported in the absence of hydrodynamics for disks (37, 38), spheres (39), and dumbbell swimmers (40). But, whereas these examples study the behavior under the influence of hard boundaries, biological swimmers typically interact with soft boundaries, such as membranes and biofilms. Another design variable is the possibility that the boundary itself is active, as in the surface of a bacterium covered with flagellae or, as demonstrated recently, active nematic vesicles (41).In this work, we propose and investigate an active matter system under flexible, active confinement. We call this system an active colloidal cell. Our realization of an active colloidal cell consists of independent particles, called spinners (42), that translate and rotate in two dimensions and are constrained within a finite area by a flexible boundary that is also built from spinners. Each spinner has a gear-like geometry, which consists of a large central disk and four smaller satellite disks (Fig. 1A). Similar gear-shaped rigid aggregates of self-propelled particles have been formed experimentally (43). Spinners are freely mobile in the cell interior. On the cellular boundary, spinners are connected to one another by a flexible chain of beads attached by finitely extensible springs. Both the interior and the boundary spinners can be subject to a clockwise or counterclockwise driving torque, which makes them active.Open in a separate windowFig. 1.Schematic of the confined spinner models. (A) The active colloidal cell is made up of spinners driven counterclockwise (blue) or clockwise (yellow). Boundary spinners are connected by a flexible bead–spring chain (gray). We compare the behavior of a continuum model (B) to a microscopic model (C). The compartmentalization of interior spinners is visualized by coloring the Voronoi tessellation in the microscopic model.Rotationally driven particles can synchronize and self-organize (44, 45) in the absence (42) and in the presence (46–48) of hydrodynamic interactions. Crystallization has recently been observed in rotating magnetic Janus colloids (49) and fast-moving bacteria (50). Spinners in the interior of the cell resemble molecular motors that push themselves forward on their neighbors and, thus, sustain convective dynamics. The effect of the boundary spinners is similar to that found in the cilia of living tissues, which stir nearby fluid. Our results demonstrate that a natural consequence of the activity present in the colloidal cell is control over both its external shape and internal structure. We report compartmentalization into regions of clockwise and counterclockwise spinners––a behavior which is affected by, and can be controlled via, properties of the enclosing boundary configuration as previously suggested (51). Transitions in the internal structure of the colloidal cell occur as its radius increases, and as the composition of the interior spinners and the patterning of the boundary are varied.A previous study of spinners in bulk (42) showed phase separation into clockwise- and counterclockwise domains. Cates and collaborators (6, 52, 53) have suggested that phase separation is a generic consequence of local energy input in an otherwise equilibrium system. Here and in the study of bulk spinners we demonstrate phase separation due to local rotational, rather than translational, energy input. We obtain our results using a particulate, microscopic model (Fig. 1C) as well as a continuum model (Fig. 1B). This allows us to conclude that the phenomena we observe are robust with respect to details of the model.In this study we use two models to study the behavior of an active colloidal cell, illustrated in Fig. 1. The microscopic model describes spinners as individual particles and simulates their motion using Langevin dynamics. It resolves the behavior of individual spinners but does not include hydrodynamic effects. In contrast, the continuum model describes the spinner system as a viscous binary fluid, which is governed by an incompressible Navier–Stokes equation coupled to a Cahn–Hilliard equation. Both models are described in detail in Materials and Methods below. Note that the microscopic model was introduced in earlier work using Brownian dynamics (42) and is extended here to include boundaries.     

Multiaddressable molecular rectangles with reversible host–guest interactions: Modulation of pH-controlled guest release and capture

A series of multiaddressable platinum(II) molecular rectangles with different rigidities and cavity sizes has been synthesized by endcapping the U-shaped diplatinum(II) terpyridine moiety with various bis-alkynyl ligands. The studies of the host–guest association with various square planar platinum(II), palladium(II), and gold(III) complexes and the related low-dimensional gold(I) complexes, most of which are potential anticancer therapeutics, have been performed. Excellent guest confinement and selectivity of the rectangular architecture have been shown. Introduction of pH-responsive functionalities to the ligand backbone generates multifunctional molecular rectangles that exhibit reversible guest release and capture on the addition of acids and bases, indicating their potential in controlled therapeutics delivery on pH modulation. The reversible host–guest interactions are found to be strongly perturbed by metal–metal and π–π interactions and to a certain extent, electrostatic interactions, giving rise to various spectroscopic changes depending on the nature of the guest molecules. Their binding mode and thermodynamic parameters have been determined by 2D NMR and van’t Hoff analysis and supported by computational study.The study of metal–metal interactions has drawn enormous attention since the past two decades because of the intriguing spectroscopic and photophysical properties arising from the close proximity of the metal centers (1, 2). Square planar d⁸ platinum(II) complexes with coordination unsaturation are one of the important classes of metal complexes that have been extensively explored because of their capability to exhibit metal–metal interactions and display rich photophysical properties (3–26). Platinum(II) terpyridine complexes have been found to exhibit rich polymorphism in the solid state (16–20) owing to their square planar coordination geometry, which permits facile access to Pt(II)···Pt(II) interactions as well as π–π interactions between the chromophores. It was not until 2001 that the first successful synthesis of platinum(II) terpyridine alkynyl complexes, which possess enhanced solubility and luminescence compared with the chloro counterpart, was reported (16). Additional efforts have been devoted to the use of the system to respond to external stimuli, such as variation in solvent composition (17, 18), pH (19, 20), temperature (21, 22), addition of ionic (24–26), and polymeric species (27, 28), in which spectral changes induced by strong Pt(II)···Pt(II) and π−π interactions have been displayed.In the past few decades, enormous efforts have been devoted to the construction of molecular architectures by fusing the organic framework to the transition metal centers through self-assembly processes (29–57). There has been continuous interest in the construction of stimuli-responsive metallosupramolecular architectures with diverse sizes, shapes, and symmetries to rationalize the criteria for molecular recognition and impart them on unique areas of applications, such as stereoselective guest encapsulation and molecular transporting devices (45–65). Although such a variety of metal–organic macrocyclic architectures has been reported, those involving the use of noncovalent interactions other than those of hydrogen bonding, donor–acceptor, electrostatic, and hydrophobic–hydrophobic interactions as well as luminescence changes that depend on the nature of the guests, which would be attractive for chemo- and biosensing, have been rare and are rather underexplored. Examples of such systems that can exhibit reversible host–guest association are also limited.Since the discovery of anticancer properties of cisplatin in 1969 (58), the coordination chemistry and the development of related species with enhanced properties and reduced cytotoxicity have received enormous attention. Although the potency and cytotoxicity studies are important, the availability of the drugs and their transport and release to the site of action are equally important. Thus, the design of smart drug delivery systems has been an area of growing interest. The first phosphorescent molecular tweezers making use of the alkynylplatinum(II) terpyridine moiety have been reported by our group to show their host–guest interactions with transition metal complexes (57). However, the opened structures of the tweezers have limited their selectivity and functionality. To accomplish the controlled drug delivery functionalities, the first main strategy is to rigidify the molecular architecture of the host from tweezers to a rectangle, so that the guest molecules would be better accommodated within the cavity, which may lead to a more selective encapsulation of guests within a definite size and steric environment. The possibility of introducing responsive functionalities into the molecular rectangles, which may serve as models for the study of on-demand controlled guest capture and release systems, has also been explored. pH-sensitive pyridine moieties have, therefore, been incorporated into the backbone of the rectangle to modulate the reversible host–guest interaction within the constrained rectangle environment on protonation/deprotonation of the pyridine nitrogen atom to achieve multiaddressable functions that would not have been readily achievable with the molecular tweezers structure. Additionally, the use of various platinum and gold complexes as guest molecules, which have been shown to display anticancer therapeutic behavior (58–65), may lead to the design of a smart multiaddressable molecular rectangle system that could capture and release specific guest molecules under different pH conditions to achieve proof-of-principle on-demand controlled drug delivery. Herein, the design and synthesis of a series of alkynylplatinum(II) terpyridine molecular rectangles (Fig. 1) with different geometries, topologies and electronic properties are reported. Moreover, the encapsulation of various guest molecules is also investigated in detail to provide a proof-of-principle model for the design of artificial drug delivery systems with the modulation of drug release by pH.Open in a separate windowFig. 1.Molecular structures of rectangles 1−4.     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.     

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.TRIM5α potently inhibits infection by HIV-1 and other retroviruses at an early postentry stage in a species-specific manner (1). Rhesus TRIM5α (rhTRIM5α) potently blocks HIV-1 infection (2). In contrast, human TRIM5α (huTRIM5α) only weakly inhibits HIV-1, but potently restricts N-tropic murine leukemia viruses (N-MLV) (3, 4). TRIM5α is able to induce premature capsid disassembly (5) and activate downstream innate immune responses upon recognizing the retroviral capsid lattice (6).TRIM5α is composed of RING, B-box 2, coiled-coil (CC), and B30.2 (PRY/SPRY) domains (Fig. 1A), similar to many tripartite/RBCC motif (TRIM) family members (7). The RING domain functions as an E3 ubiquitin ligase (8); the B-box 2 domain mediates formation of higher-order structure and self-association (9, 10); and the coiled-coil domain mediates dimerization (11) and facilitates the formation of the hexagonal lattice (12). The PRY/SPRY domain is essential for recognition of retroviral capsids and determines the specificity of restriction (13, 14). Two linker regions, L1 and L2, separate RING/B-box 2 and coiled-coil/(PRY/SPRY) domains, respectively (Fig. 1A). The antiviral potency of TRIM5α has been shown to correlate with its affinity for the viral capsid lattice (5). Interestingly, a single amino acid change from arginine to proline at residue 332 (R332P) in the PRY/SPRY domain of huTRIM5α conferred the ability to restrict HIV-1 (14–16).Open in a separate windowFig. 1.(A) Schematic depiction of dimeric TRIM5α. The four domains are colored differently and their respective molecular masses are indicated. The two linker regions are labeled L1 and L2. (B) Size-exclusion chromatograms of rhesus (solid) and human (dashed) MBP-TRIM5α PRY/SPRY. The molecular masses of protein standards are indicated at the top.HIV-1 capsid (CA) proteins can assemble into closed fullerene cones or helical tubes; other structurally homologous retrovirus CA proteins form cylindrical or spherical capsids (17–19). Despite the diverse array of retroviral capsids, different shapes are recognized by the same TRIM5α protein or highly homologous orthologs (20). The binding interaction requires an assembled capsid lattice as individual CA molecules do not have an appreciable affinity to TRIM5α (21). This broad, yet specific lattice pattern-sensing ability resides in the capsid-recognition PRY/SPRY domain of TRIM5α (5, 13, 14, 16, 22). A TRIM5α truncation construct containing the coiled-coil and the PRY/SPRY domains (CC-SPRY) is sufficient to bind and disrupt HIV-1 CA assembly (23). However, the lack of detailed structural data on TRIM5α PRY/SPRY poses an obstacle to understanding the pattern-sensing mechanism by which TRIM5α interacts with the retrovirus capsid. Here we report the crystal structure of the rhTRIM5α PRY/SPRY domain that reveals important features for viral capsid recognition. Both our EM and biochemical data demonstrate that rhTRIM5α PRY/SPRY (PRY/SPRY_rh) alone is able to recognize HIV-1 capsid tubes, whereas huTRIM5α PRY/SPRY (PRY/SPRY_hu) cannot. These findings provide a structural framework that enables us to begin understanding the capsid pattern-recognition mechanisms of TRIM5α.     

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.Amyloidosis refers to a group of protein structural disorders characterized by the extracellular deposits of insoluble amyloid fibrils resulting from abnormal conformational changes (1–5). Amyloid fibrils have a characteristic ultrastructural appearance and a β-pleated sheet core structure that consists of full-length proteins and/or fragments of either WT or mutant proteins found in familial diseases (2, 6–8). In humans, 28 amyloidogenic proteins have been identified. They are associated with prominent diseases such as Alzheimer’s disease, hemodialysis-associated amyloidosis, and familial amyloid polyneuropathy (2, 9, 10). To develop a therapeutic strategy for these disorders, it is essential to understand the mechanisms of amyloid fibril formation. Currently, the molecular and biological mechanisms that convert proteins into amyloid fibrils in vivo and in vitro remain largely unknown.Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in human and mouse plasma high-density lipoproteins (HDLs) (11) and the most important protein associated with murine senile amyloidosis because it is the precursor of amyloid fibrils (AApoAII) (12–15). Seven alleles of the apoA-II gene have been found among inbred strains of mice, with polymorphisms in 15 nucleotide positions comprising eight amino acid positions (16). Each inbred laboratory mouse strain has a single type apoA-II protein, and the pathological findings of senile amyloidosis in strains with type A, B, or C apoA-II (APOA2A, APOA2B, or APOA2C, respectively) have been investigated (13, 17, 18). C57BL/6J, ICR, and DBA/2 strains have APOA2A and exhibit a moderate incidence of mild amyloid deposits with aging (19, 20). BALB/c, C3H/He, N2B, 129/SV, and SAMR1 strains have APOA2B and exhibit a low incidence of slight amyloid deposits with aging. In contrast, the SAMP1 strain has APOA2C and spontaneously exhibits a high incidence of severe systemic amyloid deposits with aging (20–22). We previously reported a unique mechanism in which N- and C-terminal peptides of apoA-II protein associated into amyloid fibrils in vitro (23) according to the nucleation-dependent polymerization model, which can explain the general mechanisms of amyloid fibril formation (24–28). The 11-residue amino acid sequence from positions 6–16 in the N terminus of apoA-II protein is critical for polymerization into amyloid fibrils. The 18-residue amino acid sequence from positions 48–65 in the C terminus of apoA-II is also necessary for nucleation, but not for the extension phase. Both sequences are common, and there is no substitution among APOA2A, APOA2B, and APOA2C (Fig. 1).Open in a separate windowFig. 1.Amino acid sequences of mouse apoA-II and synthetic partial peptides. For types A, B, and C apoA-II proteins (APOA2A, APOA2B, and APOA2C, respectively), the two amino acid sequences indicated in the red-colored boxes at positions 6–16 at the N terminus and 48–65 at the C terminus are the essential and common sequences required for amyloid fibril formation (23). Synthetic partial peptides were used to evaluate polymerization into amyloid fibrils in vitro and suppression against amyloid deposition in mice. The bold and blue-colored letters at positions 9, 16, 54, and 62 indicate the four variant amino acids in the core sequences for types A/B/C and F apoA-II proteins. Peptides containing orange letters represent substitutions of the a48/65(N62K) peptide.We hypothesized that some amino acid substitutions in these N- and C-terminal amyloidogenic sequences of apoA-II might inhibit the polymerization of apoA-II into amyloid fibrils. In that regard, type F apoA-II (APOA2F) contains four substitutions in the N- and C-terminal peptides relative to APOA2C (16) (Fig. 1). In this study, we evaluated the in vivo incidence of amyloidosis in mice having APOA2F and compared it with those in mice having APOA2A or APOA2C. We also analyzed the ability of APOA2F peptides to polymerize into amyloid fibrils in vitro. In previous studies, we found that injection of a very small amount of AApoAII amyloid fibrils markedly accelerated amyloid deposition (13–15). We demonstrated that mice with APOA2F were absolutely resistant against senile amyloidosis, even after induction of amyloidosis by injection with type C AApoAII fibrils. Thus, we have succeeded in suppressing amyloid deposits in amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We thus demonstrate that the C-terminal sequence of APOA2F is an important inhibitor of polymerization into amyloid fibrils in vitro and in vivo. These findings provide a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro.     

Robust efficiency and actuator saturation explain healthy heart rate control and variability

The correlation of healthy states with heart rate variability (HRV) using time series analyses is well documented. Whereas these studies note the accepted proximal role of autonomic nervous system balance in HRV patterns, the responsible deeper physiological, clinically relevant mechanisms have not been fully explained. Using mathematical tools from control theory, we combine mechanistic models of basic physiology with experimental exercise data from healthy human subjects to explain causal relationships among states of stress vs. health, HR control, and HRV, and more importantly, the physiologic requirements and constraints underlying these relationships. Nonlinear dynamics play an important explanatory role––most fundamentally in the actuator saturations arising from unavoidable tradeoffs in robust homeostasis and metabolic efficiency. These results are grounded in domain-specific mechanisms, tradeoffs, and constraints, but they also illustrate important, universal properties of complex systems. We show that the study of complex biological phenomena like HRV requires a framework which facilitates inclusion of diverse domain specifics (e.g., due to physiology, evolution, and measurement technology) in addition to general theories of efficiency, robustness, feedback, dynamics, and supporting mathematical tools.Biological systems display a variety of well-known rhythms in physiological signals (1–6), with particular patterns of variability associated with a healthy state (2–6). Decades of research demonstrate that heart rate (HR) in healthy humans has high variability, and loss of this high HR variability (HRV) is correlated with adverse states such as stress, fatigue, physiologic senescence, or disease (6–13). The dominant approach to analysis of HRV has been to focus on statistics and patterns in HR time series that have been interpreted as fractal, chaotic, scale-free, critical, etc. (6–17). The appeal of time series analysis is understandable as it puts HRV in the context of a broad and popular approach to complex systems (5, 18), all while requiring minimal attention to domain-specific (e.g., physiological) details. However, despite intense research activity in this area, there is limited consensus regarding causation or mechanism and minimal clinical application of the observed phenomena (10). This paper takes a completely different approach, aiming for more fundamental rigor (19–24) and methods that have the potential for clinical relevance. Here we use and model data from experimental studies of exercising healthy athletes, to add simple physiological explanations for the largest source of HRV and its changes during exercise. We also present methods that can be used to systematically pursue further explanations about HRV that can generalize to less healthy subjects.Fig. 1 shows the type of HR data analyzed, collected from healthy young athletes (n = 5). The data display responses to changes in muscle work rate on a stationary bicycle during mostly aerobic exercise. Fig. 1A shows three separate exercise sessions with identical workload fluctuations about three different means. With proper sleep, hydration, nutrition, and prevention from overheating, trained athletes can maintain the highest workload in Fig. 1 for hours and the lower and middle levels almost indefinitely. This ability requires robust efficiency: High workloads are sustained while robustly maintaining metabolic homeostasis, a particularly challenging goal in the case of the relatively large, metabolically demanding, and fragile human brain.Open in a separate windowFig. 1.HR responses to simple changes in muscle work rate on a stationary bicycle: Each experimental subject performed separate stationary cycle exercises of ∼10 min for each workload profile, with different means but nearly identical square wave fluctuations around the mean. A typical result is shown from subject 1 for three workload profiles with time on the horizontal axis (zoomed in to focus on a 6-min window). (A) HR (red) and workload (blue); linear local piecewise static fits (black) with different parameters for each exercise. The workload units (most strenuous exercise on top of graph) are shifted and scaled so that the blue curves are also the best global linear fit. (B) Corresponding dynamics fits, either local piecewise linear (black) or global linear (blue). Note that, on all time scales, mean HR increases and variability (HRV) goes down with the increasing workload. Breathing was spontaneous (not controlled).Whereas mean HR in Fig. 1A increases monotonically with workloads, both slow and fast fluctuations (i.e., HRV) in HR are saturating nonlinear functions of workloads, meaning that both high- and low-frequency HRV component goes down. Results from all subjects showed qualitatively similar nonlinearities (SI Appendix). We will argue that this saturating nonlinearity is the simplest and most fundamental example of change in HRV in response to stressors (11, 12, 25) [exercise in the experimental case, but in general also fatigue, dehydration, trauma, infection, even fear and anxiety (6–9, 11, 12, 25)].Physiologists have correlated HRV and autonomic tone (7, 11, 12, 14), and the (im)balance between sympathetic stimulation and parasympathetic withdrawal (12, 26–28). The alternation in autonomic control of HR (more sympathetic and less parasympathetic tone during exercise) serves as an obvious proximate cause for how the HRV changes as shown in Fig. 1, but the ultimate question remains as to why the system is implemented this way. It could be an evolutionary accident, or could follow from hard physiologic tradeoff requirements on cardiovascular control, as work in other systems suggests (1). Here, the explanation of HRV similarly involves hard physiological tradeoffs in robust efficiency and employs the mathematical tools necessary to make this explanation rigorous in the context of large measurement and modeling uncertainties.