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Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer(1–6). However, numerous viruses and mobile genetic elements encode parts of the translational apparatus(7–9), potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of three of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have selective advantage over wild organisms due to virus-resistance, we also repurpose a third codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature(10). Our results suggest a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.

T-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-box riboswitches is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches, and de novo prediction of binding specificities requires advanced knowledge of computational tools to annotate riboswitch secondary structure features. Here, we present the T-box Riboswitch Annotation Database (TBDB, https://tbdb.io), an open-access database with a collection of 23,535 T-box riboswitch sequences, spanning the major phyla of 3,632 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory targets. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employ apidaecin-like, antimicrobial peptides observed to temporarily sequester and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent matter, these peptides granted a conditional lambda phage resistance to a recoded

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity.

AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells–Dawson structure K6[P2Mo18O62] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.

Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein–protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox–Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based onin vitrooptimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6–DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 MNaNO3, 0.03 MNa2HPO4, 0.03 MNH2SO4, 0.1 MMES/imidazole pH 6.5. The data set was indexed and merged in space groupC2221, with unit-cell parametersa= 75.74,b= 165.59,c= 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

Highly selective molecular binding and the subsequent dynamic protein assemblies control the adhesion of mammalian cells. Molecules that inhibit cell adhesion have the therapeutic potential for a wide range of diseases. Here, we report an efficient synthesis (2–4 steps) of a class of squaramide molecules that mimics the natural tripeptide ligand Arg-Gly-Asp (RGD) that mediates mammalian cell adhesion through binding with membrane protein integrin. In solution, this class of squaramides exhibits a higher potency at inhibiting mammalian cell adhesion than RGD tripeptides. When immobilized on a bio-inert background formed by self-assembled monolayers of alkanethiols on gold films, squaramide ligands mediate vastly different intracellular structures than RGD ligands. Immunostaining revealed that the focal adhesions are smaller, but with a larger quantity, for cells adhered on squaramides than that on RGD ligands. Furthermore, the actin filaments are also more fibrous and well distributed for cell adhesion mediated by squaramide than that by RGD ligands. Quantification reveal that squaramide ligands mediate about 1.5 times more total focal adhesion (measured by the summation of the area of all focal adhesions) than that by natural RGD ligands. This result suggests that cell adhesion inhibitors, while blocking the attachment of cells to surfaces, may induce more focal adhesion proteins. Finally, this work demonstrates that immobilizing new ligands on bioinert surfaces provide a powerful tool to study mammalian cell adhesion.

The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo- lymphangiogenesis.

Smad proteins form multimeric complexes consisting of the ‘common partner’ Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-b and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein–protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-beta-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.

We report a structural motif based on a C(3)-symmetric bowl-shaped core, on which three substituted amino acids on the periphery adopt either a folded or a spread-out conformation. This class of chiral folded structures is achieved by controlling the reactivity of the stereogenic protons on the nonplanar aromatic rings of trioxatricornan to afford predominantly C(3)-symmetric isomers. Bromination of trioxatricornan afforded a C(1)-symmetric and a C(3)-symmetric trisubstituted isomer, with the former being the major product as a statistical consequence during the reaction cascade. To obtain the C(3) symmetric isomer as the major product, C-H activation by means of ortho-lithiation with the bulky tert-butyl lithium and tetramethylethylenediamine was followed by a nucleophilic substitution that successfully reversed the statistically controlled regioselectivity. Further derivatization of the trioxatricornan with amino acids or menthol afforded diastereomers that were resolved by preparative chromatography. The absolute configurations of the diastereomers were determined by vibrational circular dichroism (VCD) in combination with density functional theory (DFT) and electronic circular dichroism (ECD). The folding structure of cysteine-derivatized trioxatricornan diastereomers was determined by two-dimensional NMR spectroscopy and molecular dynamics calculation, which revealed that one diastereomer has the amino acids folded toward the cavity of trioxatricornan and the other has a "spread-out" structure.

Ever since point groups of symmetry have been used to describe molecules after Van't Hoff and Le Bel proposed tetrahedral structures for carbon atoms in 1874, it remains difficult to design chiral molecules with polyhedral symmetry T, O, or I. Past theoretical and experimental studies have mainly accomplished molecular structures that have the conformations for satisfying the T symmetry. In this work, we present a general theoretical approach to construct rigid molecular structures that have permanently the symmetry of T, O, and I. This approach involves desymmetrizaton of the vertices or the edges of Platonic solid-shaped molecules with dissymmetric moieties. Using density functional theory (DFT) and assisted model building and energy refinement (AMBER) computational methods, the structure, the rigidity, and the symmetry of the molecule are confirmed by assessing the lowest energy conformation of the molecule, which is initially presented in a planar graph. This method successfully builds molecular structures that have the symmetry of T, O, and I. Interestingly, desymmetrization of the edges has a more stringent requirement of rigidity than desymmetrization of the vertices for affording the T, O, or I symmetry. Chirality, 2008. © 2008 Wiley-Liss, Inc.

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001, eLife digest Many scientists use ‘model’ species—such as the fruit fly or a nematode worm called Caenorhabditis elegans—in their research because these organisms have useful features that make it easier to carry out many experiments. For example, C. elegans has a smaller genome compared to many other animals, which is useful for studying the roles of individual genes or stretches of DNA. Transcription factors are a type of protein that can bind to specific stretches of DNA and help to switch certain genes on or off. These ‘motifs’ may be close to the gene or further away in the genome, and therefore, must stand out amongst the rest of the DNA, like lights on a landing strip. However, the motifs for only 10% of the estimated 763 transcription factors in C. elegans have been identified so far. In this study, Narasimhan, Lambert, Yang et al. used a technique called a ‘protein binding microarray’ to identify the motifs for many more of the C. elegans transcription factors. These findings were then used to predict motifs for other transcription factors. Together, these methods increased the proportion of C. elegans transcription factors with known DNA-binding motifs from 10% to around 40%. Now that more DNA motifs have been identified, it is possible to look for similarities and differences between them. For example, Narasimhan, Lambert, Yang et al. found that transcription factors with similar sequences can bind to very varied motifs. On the other hand, some transcription factors that are very different are able to recognize very similar motifs. The experiments also indicate that motifs found very close to genes—in sequences known as ‘promoters’—may be able to interact with many proteins to influence the activity of genes. Narasimhan, Lambert, Yang et al.'s findings increase the number of C. elegans transcription factors with a motif, bringing the knowledge of these proteins more in line with the better-studied transcription factors of humans and fruit flies. The next challenge is to identify DNA motifs for the remaining 60% of transcription factors. DOI: http://dx.doi.org/10.7554/eLife.06967.002

Background: SOX transcription factors constitute an attractive target class for intervention with small molecules as they play a prominent role in the field of regenerative biomedicine and cancer biology. However, rationally engineering specific inhibitors that interfere with transcription factor DNA interfaces continues to be a monumental challenge in the field of transcription factor chemical biology. Polyoxometalates (POMs) are inorganic compounds that were previously shown to target the high-mobility group (HMG) of SOX proteins at nanomolar concentrations. In continuation of this work, we carried out an assessment of the selectivity of a panel of newly synthesized organo-polyoxometalate hybrids in targeting different transcription factor families to enable the usage of polyoxometalates as specific SOX transcription factor drugs. Results: The residual DNA-binding activities of 15 different transcription factors were measured after treatment with a panel of diverse polyoxometalates. Polyoxometalates belonging to the Dawson structural class were found to be more potent inhibitors than the Keggin class. Further, organically modified Dawson polyoxometalates were found to be the most potent in inhibiting transcription factor DNA binding activity. The size of the polyoxometalates and its derivitization were found to be the key determinants of their potency. Conclusion: Polyoxometalates are highly potent, nanomolar range inhibitors of the DNA binding activity of the Sox-HMG family. However, binding assays involving a limited subset of structurally diverse polyoxometalates revealed a low selectivity profile against different transcription factor families. Further progress in achieving selectivity and deciphering structure-activity relationship of POMs require the identification of POM binding sites on transcription factors using elaborate approaches like X-ray crystallography and multidimensional NMR. In summary, our report reaffirms that transcription factors are challenging molecular architectures and that future polyoxometalate chemistry must consider further modification strategies, to address the substantial challenges involved in achieving target selectivity.

It has recently been proposed that the sequence preferences of DNA-binding TFs (transcription factors) can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. In the present study we examine the crystal structure of the HMG (high-mobility group) domain of Sox4 [Sry (sex-determining region on the Y chromosome)-related HMG box 4] bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2, we observed subtle conformational differences at the DNA-binding interface. Furthermore, using quantitative electrophoretic mobility-shift assays we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These results suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adaptations lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

Aberrant expression of transcription factors is a frequent cause of disease, yet drugs that modulate transcription factor protein-DNA interactions are presently unavailable. To this end, the chemical tractability of the DNA binding domain of the stem cell inducer and oncogene Sox2 was explored in a high-throughput fluorescence anisotropy screen. The screening revealed a Dawson polyoxometalate (K(6)[P(2)Mo(18)O(62)]) as a direct and nanomolar inhibitor of the DNA binding activity of Sox2. The Dawson polyoxometalate (Dawson-POM) was found to be selective for Sox2 and related Sox-HMG family members when compared to unrelated paired and zinc finger DNA binding domains. [(15)N,(1)H]-Transverse relaxation optimized spectroscopy (TROSY) experiments coupled with docking studies suggest an interaction site of the POM on the Sox2 surface that enabled the rationalization of its inhibitory activity. The unconventional molecular scaffold of the Dawson-POM and its inhibitory mode provides strategies for the development of drugs that modulate transcription factors.

This work studies the phase separations between polymers and a small molecule in a common aqueous solution that do not have well-defined hydrophobic-hydrophilic separation. In addition to poly(acrylamide) (PAAm) and poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP) also promotes liquid crystal (LC) droplet formation by disodium cromoglycate (5'DSCG) solvated in water. In the presence of these polymers, the concentration of 5'DSCG needed for forming LC droplets is substantially lower than that needed for forming an LC phase by 5'DSCG alone. To define the concentration ranges that 5'DSCG molecules form liquid crystals (either as droplets or as an isotropic-LC mixture), we constructed ternary phase diagrams for 5'DSCG, water, and a polymer - PVA, PVP, or PAAm. We discovered that PAAm with high molecular weight promotes LC droplet formation by 5'DSCG more effectively than PAAm with low molecular weight. At the same weight percentage, long-chain PAAm can cause 5'DSCG to form LC droplets in water, whereas short-chain PAAm does not. Poly(vinyl pyrrolidone) (PVP), which has functional groups that are more dissimilar to 5'DSCG than PVA and PAAm, promotes LC droplet formation by 5'DSCG more effectively than either of the other two polymers. Additionally, small angle neutron scattering data revealed that the assembly structure of 5'DSCG promoted by the presence of PVA is similar to the thread structure formed by 5'DSCG alone. Together, these results reveal how noncovalent polymerization can be promoted by mixing thermodynamically incompatible molecules and elucidate the basic knowledge of nonamphiphilic colloidal science.

Immobilizing peptides or proteins on bioinert surfaces enables the elucidation of ligand−receptor interaction in complex biological systems. Here, we report a highly chemoselective surface reaction that immobilizes peptides exclusively via N-terminus cysteine residue in a peptide. At pH 5.5, only N-terminus cysteines of peptides couple covalently with phenoxy amino squarate moieties presented on self-assembled monolayers (SAMs) of alkanethiols on gold films. The selectivity of this surface reaction can tolerate the presence of internal cysteines in close proximity to basic residues such as histidines. We demonstrated this selective surface reaction by mammalian cell adhesion and by SAMDI mass spectroscopy of the SAMs.

Crosslinked silica aerogel (x-aerogel) is low density nanostructured porous material with good mechanical properties. The mechanical characterization tests of x-aerogel were performed to establish the feasibility of using this material in different engineering applications as a multi-functional material. Mechanical response of x-aerogel was studied under compression, three-point bending, tension and DMA (dynamic mechanical analysis) tests. A primary glass transition temperature of 130 °C was identified for xaerogel material through DMA. Under uniaxial compression, x-aerogel was found linearly elastic under small strains (

The compressive failure of continuous fiber reinforced composites has received considerable attention in the recent years because it typically occurs at a stress level that is 40-50 % below the tensile strength of the composite. This paper is primarily focused on enhancing the compressive strength of polymer matrix composites (PMC) through a series of modifications in material composition and thermoplastic pultrusion manufacturing process variables. Low-cost commodity resins such as polypropylene (PP), suffer primarily due to low compressive strength. Enhancement of the compressive strength of pultruded thermoplastic composites is achieved by improving the yield strength of the surrounding matrix in shear and reducing fiber misalignment in the composite through optimization of manufacturing process variables. The dispersed platelets are typically one micron in length but only a nanometer in thickness. A single-screw extruder was used to facilitate nanoclay dispersion in PP. This new family of materials exhibits enhanced stiffness and strength of the matrix material, through the inclusion of exfoliated nano-scale montmorillonite particles in the fabrication of resin pre-impregnated (prepreg) glass fiber filaments. After the prepreg was pultruded to form a composite laminate, uniaxial compression tests were performed to determine compression strength of the laminate. Scanning Electron Micrographs (SEM) were taken to examine the failure surfaces. Transmission Electron Microscopy (TEM), were also employed to reveal all nano-scale platelet morphologies, namely exfoliated, intercalated and stacked structures within the samples. Dramatic improvement in compressive strength and compressive modulus were observed with relatively low nanoclay loadings. Compressive strength tests were also performed on aged pultruded PP composite with 0% and 1 % nanoclay loadings, and the data were compared with compressive strength data for unaged specimens. A significant increase (12-20%) in compressive strength was observed after the specimens have been aged for 15 months under ambient conditions. However, a 30% decrease in compressive modulus due to aging was recorded for specimens with 1% clay loading. Multi-scale simulations of nanoclay/polymer interface behavior are currently in progress in order to understand the strength enhancement mechanism.