There is currently a tremendous interest in developing bioorthogonal “click chemistry” methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels−Alder reactions have ...received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels−Alder reaction. We synthesized four different building blocks for 3′-terminal, 5′-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels−Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site‐specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and ...trans‐cyclooctene units that react with tetrazine derivatives in an inverse‐electron‐demand Diels–Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide–cyclooctyne click reaction (right).
Promotion of cell adhesion on biomaterials is crucial for the long‐term success of a titanium implant. Herein a novel concept is highlighted combining very stable and affine titanium surface adhesive ...properties with specific cell binding moieties in one molecule. A peptide containing l‐3,4‐dihydroxyphenylalanine was synthesized and affinity to titanium was investigated. Modification with a cyclic RGD peptide and a heparin binding peptide (HBP) was realized by an efficient on‐resin combination of Diels–Alder reaction with inverse electron demand and CuI catalyzed azide–alkyne cycloaddition. The peptide was fluorescently labeled by thiol Michael addition. Conjugating the cyclic RGD and HBP in one peptide gave improved spreading, proliferation, viability, and the formation of well‐developed actin cytoskeleton and focal contacts of osteoblast‐like cells.
Let′s stick together: An efficient on‐resin combination of a Diels–Alder reaction with inverse electron demand and a CuI catalyzed azide–alkyne cycloaddition is used to link a stable and affine TiO2‐binding peptide to two cell adhesive motifs. The multifunctional construct improved the cell adhesion of osteoblast‐like cells through the cooperative action of the peptides. DOPA=l‐3,4‐dihydroxylphenylalanine.
Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that ...features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.
DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA ...methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report ...on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.
Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using 3H-labeled ellipticine, we ...observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by 32P-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The 32P-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.
Sprague–Dawley rats were treated by intratracheal instillation with a single dose of 0.2 mg/kg body wt of 3-nitrobenzanthrone (3-NBA), and whole blood, lungs, pancreases, kidneys, urinary bladders, ...hearts, small intestines and livers were removed at various times after administration. At five posttreatment times (2 days, 2, 10, 20 and 36 weeks), DNA adducts were analysed in each tissue by 32P-postlabelling to study their long-term persistence. 3-NBA-derived DNA adducts consisting of the same adduct pattern were observed in all tissues from animals killed between 2 days and 36 weeks and between 2 days and 20 weeks in blood. DNA isolated from whole blood contained the same 3-NBA-specific adduct pattern as that found in tissues. Although total adduct levels in the blood were much lower than those found in the lung, the target organ of 3-NBA tumourigenicity, they were related (20–25%, R2 = 0.98) to the levels found in lung. In all organs, total adduct levels decreased over time to 20–30% of the initial levels till the latest time point (36 weeks) and showed a biphasic profile, with a rapid loss during the first 2 weeks followed by a much slower decline that reached a stable plateau at 20 weeks after treatment. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in target and non-target organs of the rat. The correlation between DNA adducts in lung and blood suggests that persistent 3-NBA–DNA adducts in the blood may be useful biomarkers for human respiratory exposure to 3-NBA.
Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine ...also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (P450). We examined rat, rabbit, and human hepatic microsomal samples for their ability to activate ellipticine. The extent of activation was determined by binding of 3H-labeled ellipticine to DNA and by analyzing DNA adducts by 32P-postlabeling. We demonstrate that cytochrome P450 of human hepatic microsomes activating ellipticine to species binding to DNA is analogous to that of rats, but not of rabbits. Most of the ellipticine activation in rat and human hepatic microsomes is attributed to P450 enzymes of the same subfamily, P450 3A1/2 and P450 3A4, respectively, while the orthologous enzyme in rabbit hepatic microsomes, P450 3A6, is much less efficient. With purified enzymes, the major role of P450 3A1 and 3A4 in ellipticine−DNA adduct formation was confirmed. We identified deoxyguanosine as the target for P450-mediated ellipticine binding to DNA using polydeoxyribonucleotides and deoxyguanosine 3‘-monophosphate. The results strongly suggest that rats are more suitable models than rabbits mimicking the metabolic activation of ellipticine in humans.
Die Verbesserung der Zelladhäsion auf einer Biomaterialoberfläche ist entscheidend fur den Langzeiterfolg eines Titanimplantats. Ein neues Konzept ist die Kombination aus sehr affiner und stabiler ...Adhäsion an Titanoberflächen mit spezifisch zellbindenden Motiven in einem Molekul. Ein l-3,4-Dihydroxyphenylalanin-haltiges Peptid wurde synthetisiert und auf seine Affinität fur Titan untersucht. Die Modifizierung mit einem cyclischen RGD-Peptid und einem Heparin-Bindepeptid (HBP) gelang mittels einer effizienten Festphasenkombination aus einer Diels-Alder-Reaktion mit inversem Elektronenbedarf und einer CuI-katalysierten Azid-Alkin-Cycloaddition. Daruber hinaus wurde das Peptid durch Thiol-Michael-Addition fluoreszenzmarkiert. Die Konjugation von RGD und HBP in einem Molekul fuhrte zu verbesserter Ausbreitung, Proliferation, Viabilität und Entstehung eines gut ausgebildeten Aktinzytoskeletts sowie fokaler Adhäsionskomplexe von Osteoblast-ähnlichen Zellen.
PDF
