Site-specific Replacement Research Articles

Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome.

Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.

Generation of isogenic allelic series of LMNA-mutated hiPSC lines using the novel and highly-efficient targeting platform, STRAIGHT-IN

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Organisation for Health Research and Development ZonMW Background Human induced pluripotent stem cells (hiPSCs) have demonstrated enormous potential for advancing in vitro disease modelling and genetically modified hiPSCs carrying disease-associated variants are particularly useful tools for this purpose. Until now, strategies for efficient and rapid integration of large DNA payloads (>10 kb) into specific genomic regions are still limited, thus precluding efficient and rapid targeting of hiPSC lines. Purpose and Methods To overcome this, the STRAIGHT-IN platform (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) was developed that combined different classes of site-specific recombinases with CRISPR-Cas9 mediated homologous recombination and allowed stringent site-specific replacement of large genomic fragments in hiPSCs. Here, we use STRAIGHT-IN to simultaneously generate a library of genetically matched hiPSC lines carrying multiple heterozygous mutations in LMNA gene. Mutations in LMNA, encoding Lamin A/C protein, have been associated with 5-10% cases of dilated cardiomyopathy (DCM) with conduction defects. Results Through detailed online searches of the LMNA database and literature, we identified and selected 11 LMNA mutations to insert into a wild-type hiPSC line based on the following criteria: (i) mutations associated with cardiac abnormalities, in terms of structural (DCM/heart failure) and arrhythmic phenotypes; (ii) mutations with known familiar/pedigree-relationship; (iii) different types of mutations (missense and nonsense). We then applied the workflow of STRAIGHT-IN consisting of: (1) replacing the entire LMNA genomic locus (33 kb) on one allele with a Landing Pad (LP) cassette containing Serine Recombinase (Bxb1) recognition sites; (2) reintroducing LMNA gene into the LP cassette through integration of Bxb1-Donor plasmids carrying the LMNA variants of interest via Bxb1 expression; (3) expressing a tyrosine recombinase (Cre) to excise the majority of the auxiliary exogenous DNA sequences. Conclusions STRAIGHT-IN allows simultaneous generation of a panel of 11 isogenic hiPSC lines carrying selected LMNA mutations rapidly (5-6 weeks), efficiently and cost-effectively, thus facilitating the production of specific mutated hiPSC lines for disease modelling and personalized medicine applications.

COMPATIBILITY ANALYSIS OF THE SOIL CALCIUM CONTENT BY MULTIFREQUENCY EC SENSORS

Nowadays in precision agriculture, the on-the-go measurement of soil nutrients is an important research topic in the sustainable nutrient management practices. Determining the nutrient content of soils and the judicious and site-specific replacement of missing mineral compounds of soil has a major impact on production costs in terms of current fertilizer prices. Soil sensors currently on the market can only determine total soil salinity. Therefore, selective soil salinity testing is only possible using laboratory methods. However, these methods are rather expensive, slow, and cumbersome. Growers often need faster and cheaper soil sampling process and immediate results. We believe that by developing measurement models of soil sensors, the data delivery process could be significantly shortened, so that measurement results could be processed and used even in real time. In this paper, we prove that electrical conductivity measurements can be a suitable tool for the determination of the selective salinity of soil. In our experiments, the calcium cation content of soil was measured by conductometry in laboratory conditions. In our model, we investigated the effect of a reasonable and well-considered variation of the measuring current frequency on the measurement output variable, i.e. the electrical conductivity (often abbreviated as EC) value. Our experiments have shown that with multi-frequency, solutions containing predetermined concentrations of Ca2+ ions, the EC obtained as an output parameter follows different functions.

Structural and Biological Features of G-Quadruplex Aptamers as Promising Inhibitors of the STAT3 Signaling Pathway.

In this paper, we investigate the structural and biological features of G-quadruplex (G4) aptamers as promising antiproliferative compounds affecting the STAT3 signalling pathway. Targeting the STAT3 protein through high-affinity ligands to reduce its levels or activity in cancer has noteworthy therapeutic potential. T40214 (STAT) [(G3C)4] is a G4 aptamer that can influence STAT3 biological outcomes in an efficient manner in several cancer cells. To explore the effects of an extra cytidine in second position and/or of single site-specific replacements of loop residues in generating aptamers that can affect the STAT3 biochemical pathway, a series of STAT and STATB [GCG2(CG3)3C] analogues containing a thymidine residue instead of cytidines was prepared. NMR, CD, UV, and PAGE data suggested that all derivatives adopt dimeric G4 structures like that of unmodified T40214 endowed with higher thermal stability, keeping the resistance in biological environments substantially unchanged, as shown by the nuclease stability assay. The antiproliferative activity of these ODNs was tested on both human prostate (DU145) and breast (MDA-MB-231) cancer cells. All derivatives showed similar antiproliferative activities on both cell lines, revealing a marked inhibition of proliferation, particularly at 72 h at 30 µM. Transcriptomic analysis aimed to evaluate STAT's and STATB's influence on the expression of many genes in MDA-MB-231 cells, suggested their potential involvement in STAT3 pathway modulation, and thus their interference in different biological processes. These data provide new tools to affect an interesting biochemical pathway and to develop novel anticancer and anti-inflammatory drugs.

Surface Site-Specific Replacement for Catalysis Selectivity Switching.

Surface atom replacement in materials without other composition/structure changes is challenging but is important for fundamental scientific research and for practical applications. In particular, for nanoparticles including nanoclusters, surface metal site-specific replacement with atomic precision has not yet been achieved. In this study, we for the first time achieved surface site-specific antigalvanic replacement with the remaining composition/structure and surface replacement-dependent selectivity in the electrocatalytic reduction of CO2. Density functional theory (DFT) calculations describe the catalysis selectivity switch induced by replacing Ag with Cu and explain why Cu replacement facilitates C2 production. Also, CO2 electroreduction to C2 on well-defined metal nanoclusters is first reported in this study.

STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

SummaryInserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.

Reprogramming Nonribosomal Peptide Synthetases for Site-Specific Insertion of α-Hydroxy Acids.

High-throughput engineering has the potential to revolutionize the customization of biosynthetic assembly lines for the sustainable production of pharmaceutically relevant natural product analogs. Here, we show that the substrate specificity of gatekeeper adenylation domains of nonribosomal peptide synthetases can be switched from an α-amino acid to an α-hydroxy acid in a single round of combinatorial mutagenesis and selection using yeast cell surface display. In addition to shedding light on how such proteins discriminate between amino and hydroxy groups, the remodeled domains function in a pathway context to produce α-hydroxy acid-containing linear peptides and cyclic depsipeptides with high efficiency. Site-specific replacement of backbone amines with oxygens by an engineered synthetase provides the means to probe and tune the activities of diverse peptide metabolites in a simple and predictable fashion.

Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition.

The teeth of sharks famously form a series of transversely organized files with a conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here, we use tomographic methods to investigate mandibular structures in several early branching ‘acanthodian’-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckel's cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

Therapeutic Genome Editing and In Vivo Delivery.

Improvements in the understanding of human genetics and its roles in disease development and prevention have led to an increased interest in therapeutic genome editing via the use of engineered nucleases. Various approaches have been explored in the past focusing on the development of an effective and safe system for sequence-specific editing. Compared to earlier nucleases such as zinc finger nuclease and transcription activator-like effector nuclease, the relatively low cost and ease of producing clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) systems have made therapeutic genome editing significantly more feasible. CRISPR/Cas9 genome editing has shown great potential to correct genetic mutations implicated in monogenic diseases and to eradicate latent or chronic viral infections in preclinical studies. Several CRISPR/Cas9-based therapeutics have reached the clinical stage, including treatments for inherited red blood cell disorders and Leber Congenital Amaurosis 10, as well as CRISPR/Cas9-edited T cells designed to target and destroy cancer cells. Further advances in therapeutic genome editing will rely on a safe and more efficient method of in vivo CRISPR/Cas9 delivery and improved efficiency of homology-directed repair for site-specific gene insertion or replacement. While other reviews have focused on one or two aspects of CRISPR/Cas9 genome editing, this review aims to provide a summary of the mechanisms of genome editing, the reasons for the emerging interest in CRISPR/Cas9 compared to other engineered nucleases, the current progress in developing CRISPR/Cas9 delivery systems, and the current preclinical and clinical applications of CRISPR/Cas9 genome editing.

Isomeric Effect of Nano-Inhibitors on Aβ40 Fibrillation at The Nano-Bio Interface.

Chemical and physical properties of nanobio interface substantially affect the conformational transitions of adjacent biomolecules. Previous studies have reported the chiral effect and charge effect of nanobio interface on the misfolding, aggregation, and fibrillation of amyloid protein. However, the isomeric effect of nanobio interface on protein/peptides amyloidosis is still unclear. Here, three isomeric nanobio interfaces were designed and fabricated based on the same sized gold nanoclusters (AuNCs) modified with 4-mercaptobenzoic acid (p-MBA), 3-mercaptobenzoic acid (m-MBA), and 2-mercaptobenzoic acid (o-MBA). Then three isomeric AuNCs were employed as models to explore the isomeric effect on the misfolding, aggregation, and fibrillation of Aβ40 at nanobio interfaces. Site-specific replacement experiments on the basis of theoretical analysis revealed the possible mechanism of Aβ40 interacting with isomeric ligands of AuNCs at the nanobio interfaces. The distance and orientation of -COOH group from the surface of AuNCs can affect the electrostatic interaction between isomeric ligands and the positively charged residues (R5, K16, and K28) of Aβ40, which may affect the inhibition efficiency of isomeric AuNCs on protein amyloidosis. Actually, the amyloid fibrillation kinetics results together with atomic force microscope (AFM) images, dynamic light scattering (DLS) results and circular dichroism (CD) spectra indeed proved that all the three isomeric AuNCs could inhibit the misfolding, aggregation and fibrillation of Aβ40 in a dose-dependent manner, and the inhibition efficiency was definitely different from each other. The inhibition efficiency of o-MBA-AuNCs was higher than that of m-MBA-AuNCs and p-MBA-AuNCs at the same dosage. These results provide an insight for isomeric effect at nanobio interfaces, and open an avenue for structure-based nanodrug design target Alzheimer's disease (AD) and even other protein conformational diseases.

Global Assessment of Substituents on the Basis of Analogue Series.

While bioisosteric replacements have been extensively investigated, comprehensive analyses of R-/functional groups have thus far been rare in medicinal chemistry. We introduce a new analysis concept for the exploration of chemical substituent space that is based upon bioactive analogue series as a source. From ∼24,000 analogue series, more than 19,000 substituents were isolated that were differently distributed. A subset of ∼400 substituent fragments occurred most frequently in different structural contexts. These substituents contained well-known R-groups as well as novel structures. Substitution site-specific replacement and network analysis revealed that chemically similar substituents preferentially occurred at given sites and identified intuitive substitution pathways that can be explored for compound design. Taken together, the results of our analysis provide new insights into substituent space and identify preferred substituents on the basis of analogue series. As a part of our study, all the data reported are made freely available.

Is there a joint lever? Identifying and ranking factors that determine GHG emissions and profitability on dairy farms in Bavaria, Germany

Farms are increasingly expected to contribute to greenhouse gas (GHG) mitigation actions to help governments to achieve GHG reduction commitments. In order to identify key mechanisms for GHG mitigation on farms, many studies use mass flow simulation or optimization models. However, by assuming “best practice” and not accounting for “real farm practices”, these models cannot predict variability between farms. In contrast, studies that include variability between farms can identify determinants that are important factors to reduce GHG emissions. From a farmer's perspective, it is often crucial that these mechanisms also increase farm profitability. The objectives of this article are (1) to explore factors that jointly affect GHG emissions and profitability of dairy farms and, (2) to assess if these factors cause synergies or trade-offs to simultaneously reduce GHG emissions and increase profitability. To assess variability between farms, we utilize detailed site- or farm-specific input variables for a large number of farms. To this end, we combined a detailed high quality dataset of 92 farms for the year 2013 in Bavaria, Germany. In relation to GHG emissions, we collected emission factors from national and international life cycle analysis databases, and applied national and site-specific GHG emission models. Our global sensitivity analysis identified five factors affecting GHG emissions per kg of fat and protein corrected milk in the following order of relative importance (i.e. proportion of farm variability explained): feed use efficiency (26%), weighted nitrogen balance (23%), site specific nitrogen emission factor (15%), milk yield (13%), and replacement rate (8%). Of these five factors, feed use efficiency and milk yield were also relatively important factors for profitability. However, milk yield is strongly interlinked with beef output, an important by-product of our sample dairy farms, and thus needs special attention when defining effective GHG reduction targets. Site-specific nitrogen emission factors cannot be influenced directly by farmers. This leaves three main determinants for farm variability between farms of GHG emissions i.e. on field nitrogen use efficiency, feed use efficiency and replacement rate. Since feed use efficiency was also identified as an important factor increasing profitability, this could be addressed by advisory services assessing synergies between profitability and GHG emissions. On field nitrogen use efficiency and replacement rate were not identified as an important factor affecting profitability and thus may be addressed by additional incentives for farmers, advisory service, or stricter regulations.

Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

Safety evaluation of myostatin-edited Meishan pigs by whole genome resequencing analyses

Genome editing technology can make specifically target genomic modifications, resulting in site specific DNA insertion, deletion or replacement in the genome of an organism. We have recently produced genetically engineered (GE) Meishan pigs containing a ZFN-edited myostatin (MSTN) loss-of-function mutation that leads to a clear “double muscle” phenotype as observed for Belgian cattle. In this study, whole genome resequencing was used as an approach to evaluate the safety risk, if any, associated with the introduction of a ZFN-edited myostatin (MSTN) loss-of-function mutation in a local pig breed, the Meishan pigs. The results of resequencing analyses show that the effective data from pigs of wild-type group and MSTN-edited GE group is greater than 99%. The 1× coverage rate is > 98%, and the 4× coverage rate is > 96%. The genetic variation on each chromosome is close to 1. From this whole genome resequencing study, our results demonstrated that 99.7% of single nucleotide polymorphisms (SNPs) are the same in the same genetic variation from both wild-type group and MSTN-edited GE group, implying genomic sequence variations are highly similar between the two groups of pigs.

Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5′-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.

Oligonucleotide analogues with cationic backbone linkages.

Their unique ability to selectively bind specific nucleic acid sequences makes oligonucleotides promising bioactive agents. However, modifications of the nucleic acid structure are an essential prerequisite for their application in vivo or even in cellulo. The oligoanionic backbone structure of oligonucleotides mainly hampers their ability to penetrate biological barriers such as cellular membranes. Hence, particular attention has been given to structural modifications of oligonucleotides which reduce their overall number of negative charges. One such approach is the site-specific replacement of the negatively charged phosphate diester linkage with alternative structural motifs which are positively charged at physiological pH, thus resulting in zwitterionic or even oligocationic backbone structures. This review provides a general overview of this concept and summarizes research on four according artificial backbone linkages: aminoalkylated phosphoramidates (and related systems), guanidinium groups, S-methylthiourea motifs, and nucleosyl amino acid (NAA)-derived modifications. The synthesis and properties of the corresponding oligonucleotide analogues are described.

Improvement of the activity of the anti-HIV-1 integrase aptamer T30175 by introducing a modified thymidine into the loops

In this paper, we report our investigations on analogues of the anti-human immunodeficiency virus type 1 (HIV-1) integrase (IN) aptamer T30175 in which the individual thymidines forming the loops were replaced by 5-hydroxymethyl-2′-deoxyuridine residues (H). Circular dichroism, nuclear magnetic resonance and gel electrophoresis investigations clearly indicated that all the modified aptamers preserve the ability to form the original 5′-5′ end-stacked head-to-head dimeric G-quadruplex structure, in which each G-quadruplex adopts a parallel arrangement and is characterized by three G-tetrads, three propeller loops and one bulge-loop. All the modified aptamers were tested in an IN inhibition LEDGF-independent assay. While the modified aptamers INTB-H13 and INTB-H17 showed IC50 values comparable with that of the parent aptamer (INTB-nat), analogues INTB-H2, INTB-H5 and, to a lesser extent, INTB-H9 showed a higher ability to inhibit the HIV IN than the unmodified aptamer. Molecular modelling studies evaluating the aptamer/HIV IN interaction highlighted the ability of the modified thymidines to establish several contacts with the target protein. All the data point to the importance of loops in the aptamer/target interaction and suggest that the site-specific replacement of loop residues with commercially available analogues can be considered a straightforward strategy to improve the biological activities of several G-quadruplex aptamers.

The “Janus face” of the thrombin binding aptamer: Investigating the anticoagulant and antiproliferative properties through straightforward chemical modifications

BackgroundThe thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative activities. Its chemico-physical and/or biological properties can be tuned by the site-specific replacement of selected residues. MethodsFour oligodeoxynucleotides (ODNs) based on the TBA sequence (5′-GGTTGGTGTGGTTGG-3′) and containing 2′-deoxyuridine (U) or 5-bromo-2′-deoxyuridine (B) residues at positions 4 or 13 have been investigated by NMR and CD techniques. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay) have been tested and compared with two further ODNs containing 5-hydroxymethyl-2′-deoxyuridine (H) residues in the same positions, previously investigated. ResultsThe CD and NMR data suggest that all the investigated ODNs are able to form G-quadruplexes strictly resembling that of TBA. The introduction of B residues in positions 4 or 13 increases the melting temperature of the modified aptamers by 7 °C. The replacement of thymidines with U in the same positions results in an enhanced anticoagulant activity compared to TBA, also at low ODN concentration. Although all ODNs show antiproliferative properties, only TBA derivatives containing H in the positions 4 and 13 lose the anticoagulant activity and remarkably preserve the antiproliferative one. ConclusionsAll ODNs have shown antiproliferative activities against two cancer cell lines but only those with U and B are endowed with anticoagulant activities similar or improved compared to TBA.General significance:The appropriate site-specific replacement of the residues in the TT loops of TBA with commercially available thymine analogues is a useful strategy either to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones.

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA).

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.