Encoding hierarchical assembly pathways of proteins with DNA

The structural and functional diversity of materials in nature depends on the controlled assembly of discrete building blocks into complex architectures via specific, multistep, hierarchical assembly pathways. Achieving similar complexity in synthetic materials through hierarchical assembly is challenging due to difficulties with defining multiple recognition areas on synthetic building blocks and controlling the sequence through which those recognition sites direct assembly. Here, we show that we can exploit the chemical anisotropy of proteins and the programmability of DNA ligands to deliberately control the hierarchical assembly of protein–DNA materials. Through DNA sequence design, we introduce orthogonal DNA interactions with disparate interaction strengths (“strong” and “weak”) onto specific geometric regions of a model protein, stable protein 1 (Sp1). We show that the spatial encoding of DNA ligands leads to highly directional assembly via strong interactions and that, by design, the first stage of assembly increases the multivalency of weak DNA–DNA interactions that give rise to an emergent second stage of assembly. Furthermore, we demonstrate that judicious DNA design not only directs assembly along a given pathway but can also direct distinct structural outcomes from a single pathway. This combination of protein surface and DNA sequence design allows us to encode the structural and chemical information necessary into building blocks to program their multistep hierarchical assembly. Our findings represent a strategy for controlling the hierarchical assembly of proteins to realize a diverse set of protein–DNA materials by design.

Hierarchical assembly is integral to the structural complexity and function of materials and systems that occur in nature. Muscle tissue (1), amyloid fibrils (2), and collagen networks (3) are all examples of highly organized supramolecular architectures that arise from bottom-up, multistep, regulated assembly processes. The well-controlled sequence of assembly steps along a given pathway and the specificity of interactions between components are critical to the observed structural complexity and diversity (4, 5). While nanoscale hierarchical assembly is prevalent and important in nature, and our ability to control the bottom-up assembly of synthetic nanoscale building blocks has been transformed over the past two decades (6–8), we are still limited in what can be programmed through hierarchical mechanisms (9, 10). This is due to difficulties in defining the number, type, and location of multiple interactions on synthetic building blocks, as well as limitations in controlling the interplay between orthogonal interactions to achieve a desired assembly pathway (11). The development of tools and strategies to program multistep assembly pathways of nanoscale building blocks would redefine how we control the bottom-up synthesis of materials and accelerate the discovery of novel structures with desirable properties and functions (12, 13). In this work, we address this gap by spatially encoding programmable interacting ligands (DNA) onto the surface of chemically addressable building blocks (proteins).Proteins are an important class of nanoscale building block because of their structural and functional roles in biology. As such, developing methods to synthetically engineer new materials from proteins is a common goal in the fields of synthetic biology, chemistry, and materials science, with diverse applications from catalysis (14) to immune evasion (15) and biological delivery (16). The chemical complexity of protein surfaces defines specific recognition between protein interfaces and is key to the hierarchical assembly processes observed in nature. However, their complex surfaces make it challenging to design protein building blocks that will transform into targeted materials by traversing an intended assembly pathway. While powerful de novo design strategies have been utilized to create proteins with predetermined interfaces and assembly outcomes (17, 18), this approach inherently deviates from the pool of naturally occurring protein building blocks that could be utilized for materials engineering. Other strategies have relied on introducing controlled molecular interactions to the surfaces of proteins ranging from metal coordination chemistries (19–21) to hydrophobic (22) and host–guest interactions (23, 24). Despite significant innovation in manipulating surface interactions through chemical modifications, less attention has been paid to designing protein building blocks that can undergo multistep assembly pathways mimicking those in nature (25–27), because it remains challenging to realize interactions that are simultaneously specific, orthogonal, and have tunable strengths. Indeed, methods to define interaction location and type on the surface of a building block, in conjunction with an understanding of how to control and regulate each interaction independently, are needed to successfully program hierarchical assembly pathways. Although a growing body of literature has examined assembly pathways in the context of protein crystal polymorphism (28, 29), the ability to design directional, multistep assembly processes remains elusive.In addition to programming the structures of protein assemblies using DNA origami templates with specific, directional interactions (16, 29–33), our group and others have shown that DNA ligands chemically tethered to the surfaces of proteins at specific locations can drive the assembly of proteins into one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) assemblies and crystals (34–45). Protein mutagenesis has been used to site-specifically encode multiple, orthogonal DNA interactions onto protein surfaces to program directional assembly (46). Furthermore, the programmable recognition properties of DNA surface ligands have been utilized to control the polymerization pathway of proteins (47). However, these examples all rely on a single assembly step to reach their target structure and do not teach us how to create more complex materials from multistep, hierarchical assembly of proteins, such as those observed in nature. Indeed, even when multistep DNA assembly was demonstrated for inorganic nanoparticles, the second assembly step could only be induced by chemical modification of the structure formed after an initial assembly step and the addition of more nanoparticle building blocks to the system (48).We hypothesized that, if we could define the specificity, strength, and spatial distribution of multiple specific DNA interactions on the surface of a protein, we would be able to synthesize protein building blocks that undergo spontaneous, programmed, multistep assembly processes. Here, by defining the chemical anisotropy of a protein’s surface via mutagenesis, we define DNA interactions spatially, that is, axially or equatorially with respect to the geometry of an anisotropic protein (Scheme 1A). Through careful DNA design, we modulate the relative interaction strengths of the axial and equatorial faces such that assembly via strong interactions in a single direction leads to an emergent, second interaction that can program assembly in an orthogonal direction (Scheme 1B). The emergence of this second interaction is a hallmark of hierarchical assembly observed in nature and is responsible for directing the assembly of proteins along specific, multistep pathways. This study focuses on articulating this concept for programming the assembly of nanoscale building blocks along specific, hierarchical pathways, rather than obtaining arbitrarily high registry in 2D and 3D protein materials.Open in a separate windowScheme 1.Design of Sp1m chemical surface and proposed hierarchical assembly schemes. (A) Native Sp1 (Left) presents multiple primary amines (lysines and N termini, blue) and no cysteines (red) on its surface. Three mutations were designed to remove two native lysines and introduce one cysteine per subunit. Due to the dodecameric structure of Sp1m, these mutations define the chemical anisotropy across the protein surface with amine residues only on the axial face and cysteines located only on the equatorial face. (B) Proposed assembly schemes for building blocks containing strong or weak surface interactions at their axial or equatorial positions. Strong interactions direct the first stage of assembly, leading to multivalency among weak interactions that direct the second stage of assembly.     

Surface modified solid lipid microparticles based on homolipids and Softisan® 142: preliminary characterization

ObjectiveTo preliminarily investigate three different lipid matrices consisting of two natural homolipids from Capra hircus (goat fat) and Bovine Spp. (tallow fat) and one semi-synthetic lipid (Softisan® 142) separately structured with Phospholipon® 90G (P90G) as potential delivery systems for poorly water soluble drugs.MethodsThe structured lipid matrices were characterized by differential scanning calorimetry (DSC) and employed to prepare solid lipid microparticles (SLMs) by the melt homogenization method using gradient concentrations of polysorbate 80 and at different emulsification times of 2, 5 and 10 min using a Silverson mixer. The SLMs were analyzed for morphology and particle size, thermal properties, stability studies and determination of injectability.ResultsThe results showed that SLM production was optimum at 5 % of lipid matrices, 1.5 % of polysorbate 80 and emulsification time of 5 min. Increase in polysorbate 80 concentrations decreased the particle size of the SLMs. The SLMs were well formed, spherical, smooth and non-porous with particle sizes in the ranges of (13.90 ± 2.10) μm - (0.09 ± 0.01) μm for SLMs produced from the structured - tallow fat; (13.40 ± 1.30) μm - (0.10 ± 0.01) μm for the structured - goat fat and (13.40±2.00) μm - (2.10± 1.00)μm for the structured Softisan® 142 lipid matrices. DSC traces showed that Softisan® 142 was the most crystalline of all three bulk matrices due to its high enthalpy (?7.962 mW/mg) while tallow fat was the least (?5.067 mW/mg) but addition of P90G to the matrices lowered their enthalpies mostly in the structured goat fat matrices. The SLMs when stored at 4-6 ° were most stable and syringeable with 27 G needle.ConclusionsThis suggests that structured goat fat matrices with the enthalpy of ?2.813 mW/mg will mostly favour drug loading of some poorly soluble drugs more than tallow fat (?4.892 mW/mg) and Softisan® 142 (?5.501 mW/mg).     

In vitro analysis of the homing properties of human lymphocytes: developmental regulation of functional receptors for high endothelial venules

Circulating lymphocytes leave the blood by binding to specialized high endothelial cells lining postcapillary venules in lymphoid organs or sites of chronic inflammations, migrating through the vessel wall into the surrounding tissue. The capacity of lymphocytes to recognize and bind to high endothelial venules (HEVs) is thus central to the overall process of lymphocyte traffic and recirculation. We show that viable human lymphocytes bind selectively to HEVs in frozen sections of normal human lymph nodes, thus defining a simple in vitro model for the study of human lymphocyte homing properties. Optimal conditions for the quantitative analysis of lymphocyte-HEV interaction are described. Furthermore, by using this assay, we demonstrate that the ability of human lymphocyte populations to bind to HEVs parallels their presumed migratory status in vivo. Thus, thymocytes and bone marrow cells, which are sessile in vivo, bind poorly to HEVs in comparison with mature circulating lymphocytes in peripheral blood or in peripheral lymphoid tissues. These results indicate that HEV-binding ability is a regulated property of mature lymphocytes and, as demonstrated previously in animal models, probably plays a fundamental role in controlling lymphocyte traffic in humans. The in vitro model of lymphocyte-HEV interaction thus provides a unique means to assay the migratory properties of normal and neoplastic human lymphocyte subsets, to analyze the role of lymphocyte traffic mechanisms in normal and pathologic inflammatory reactions, and to define some of the molecular mechanisms responsible for the control of lymphocyte migration and positioning in humans.     

Abaloparatide at the Same Dose Has the Same Effects on Bone as PTH (1-34) in Mice

Abaloparatide, a novel analog of parathyroid hormone-related protein (PTHrP 1–34), became in 2017 the second osteoanabolic therapy for the treatment of osteoporosis. This study aims to compare the effects of PTH (1-34), PTHrP (1-36), and abaloparatide on bone remodeling in male mice. Intermittent daily subcutaneous injections of 80 μg/kg/d were administered to 4-month-old C57Bl/6J male mice for 6 weeks. During treatment, mice were followed by DXA-Piximus to assess changes in bone mineral density (BMD) in the whole body, femur, and tibia. At either 4 or 18 hours after the final injection, femurs were harvested for μCT analyses and histomorphometry, sera were assayed for bone turnover marker levels, and tibias were separated into cortical, trabecular, and bone marrow fractions for gene expression analyses. Our results showed that, compared with PTH (1-34), abaloparatide resulted in a similar increase in BMD at all sites, whereas no changes were found with PTHrP (1-36). With both PTH (1-34) and abaloparatide, μCT and histomorphometry analyses revealed similar increases in bone volume associated with an increased trabecular thickness, in bone formation rate as shown by P1NP serum level and in vivo double labeling, and in bone resorption as shown by CTX levels and osteoclast number. Gene expression analyses of trabecular and cortical bone showed that PTH (1-34) and abaloparatide led to different actions in osteoblast differentiation and activity, with increased Runx2, Col1A1, Alpl, Bsp, Ocn, Sost, Rankl/Opg, and c-fos at different time points. Abaloparatide seems to generate a faster response on osteoblastic gene expression than PTH (1-34). Taken together, abaloparatide at the same dose is as effective as PTH (1-34) as an osteoanabolic, with an increase in bone formation but also an increase in bone resorption in male mice. © 2019 American Society for Bone and Mineral Research.     

Cocaine-seeking produced by experimenter-administered drug injections: dose-effect relationships in rats

Rationale: Relapse to drug taking is a major obstacle to the effective treatment of cocaine abuse. Animal studies have determined that various drugs are able to reinstate extinguished drug-taking behavior. Objectives: This study was designed to determine whether there is specificity in the ability of drugs to lead to cocaine-seeking and to compare potency and efficacy of a variety of drug primes. Another objective was to compare the effect of drugs with a primary dopaminergic mechanism with those having a secondary effect on dopaminergic substrates. Methods: Following acquisition of cocaine self-administration, the ability of injections of cocaine (5.0–20.0 mg/kg), amphetamine (0.30–3.0 mg/kg), methylphenidate (2.0–20.0 mg/kg), nicotine (0.0375–0.60 mg/kg), caffeine (1.25–20.0 mg/kg), morphine (0.10–10.0 mg/kg) or ^Δ9THC (0.3–3.0 mg/kg) to reinstate extinguished drug taking was measured. Tests were conducted in a single day and were comprised of three phases. The first phase consisted of a 60-min period of cocaine self-administration. During phase 2, the cocaine solution was replaced with saline and responding was extinguished during the next 3-h period. During phase 3, in which saline again was the only solution available for self-administration, responding was monitored for 3–8 h following an injection of a drug prime. Results: Reinstatement was produced by experimenter-administered injections of cocaine, amphetamine, methylphenidate and caffeine but not nicotine, morphine or ^Δ9THC. The potency and efficacy of cocaine, methylphenidate and caffeine were comparable, whereas amphetamine was more potent and efficacious. Cocaine seeking occurred primarily during the first hour following the injection. Conclusions: These findings suggest that cocaine seeking is only produced following administration of specific drugs. It is suggested that effective drug primes are those that produce a discriminative stimulus that generalizes to the stimulus produced by the reinforcing effects of cocaine. Received: 13 February 1999 / Final version: 25 June 1999