ABSTRACT
Chemical inducers of dimerization (CIDs) are cell-permeable small molecules capable of dimerizing two protein targets. The most widely used CID, the natural product rapamycin and its relatives, is immunosuppressive due to interactions with endogenous targets and thus has limited utility in vivo. Here we report a new biocompatible CID, Tmp-SLF, which dimerizes E. coli DHFR and FKBP and has no endogenous mammalian targets that would lead to unwanted in vivo side effects. We employed Tmp-SLF to modulate gene expression in a yeast three-hybrid assay. Finally, we engineered the Golgi-resident glycosyltransferase FucT7 for tunable control by Tmp-SLF in mammalian cells.
Subject(s)
Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Glycosylation , Molecular Structure , Protein Binding , Protein Multimerization , Saccharomyces cerevisiae/geneticsABSTRACT
The challenge of engineering glycosylation has been confronted by synthetic chemists, biochemists and cell biologists, each with the primary goal of optimizing glycoconjugates for therapeutic applications. In nature, glycans are constructed by glycosyltransferases that are organized in an assembly line in the endoplasmic reticulum and Golgi compartment. Recent insights into the domain architecture, localization and regulation of glycosyltransferases have provided a platform for engineering their position within the secretory pathway and access to substrates. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. These efforts epitomize the emerging field of 'synthetic glycobiology'.
Subject(s)
Glycoproteins/biosynthesis , Golgi Apparatus/metabolism , Glycoproteins/genetics , Humans , Pichia/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
We developed a polymer coating for carbon nanotubes (CNTs) that mimics the mucin glycoprotein coating of mammalian cells. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells.
Subject(s)
Cells/metabolism , Molecular Mimicry , Mucins , Nanotubes, Carbon/chemistry , Receptors, Cell Surface/metabolism , Animals , Biocompatible Materials , Biomimetic Materials/chemical synthesis , Biomimetic Materials/pharmacology , Cells/chemistry , Coated Materials, Biocompatible , Humans , Nanotechnology , Polymers , Protein BindingABSTRACT
Nucleic acid template-directed synthesis represents a powerful method for the encoded synthesis of new bioconjugates. Our laboratory previously reported a strategy for the synthesis of a new metal-DNA hybrid, metallosalen-DNA, by the DNA or RNA template-directed cross-linking of two salicylaldehyde-modified DNA oligonucleotides. The current manuscript describes the optimal assembly requirements and biophysical characterization of metallosalen-DNA conjugates containing nickel and manganese ions. Competitive assembly reactions demonstrated the template-directed nature of metallosalen-DNA formation. A single metallosalen-DNA conjugate was assembled selectively in the presence of two pairs of salicylaldehyde precursor strands and a single DNA template. Assembly reactions were sensitive to base pair mismatches in the pairing arms. Single base mismatches resulted in a loss of metallosalen-DNA conjugate yield. Metallosalen-DNA assembly yields depended on the identity and length of the template spacer, the reaction pH, and the type of metal and diamine utilized in the assembly reaction. Metallosalen-DNA conjugates were stable to a variety of conditions, including extended incubation at 50 degrees C. Nickel metallosalen-DNA remained unchanged after incubation at 80 degrees C for 24 h, while decomposition of manganese metallosalen-DNA was observed under the same conditions. Circular dichroism (CD) spectroscopy indicated that DNA duplexes containing internal metallosalen moieties adopted B-form double helices. UV thermal denaturation analysis demonstrated that 32-nucleotide duplexes containing internal metallosalen modifications displayed melting temperatures approximately 5 degrees C less than unmodified DNA duplexes.
Subject(s)
Chelating Agents/chemical synthesis , DNA/chemistry , Organometallic Compounds/chemical synthesis , Templates, Genetic , Aldehydes/chemistry , Base Pair Mismatch , Base Sequence , Circular Dichroism , Cross-Linking Reagents , Ethylenediamines/chemistry , Hydrogen-Ion Concentration , Manganese/chemistry , Nickel/chemistry , Nucleic Acid Denaturation , Oligonucleotides/chemical synthesis , RNA/chemistry , TemperatureABSTRACT
Ni-salen-DNA conjugates, prepared by template-directed synthesis, targeted oxidative adduct formation and strand scission at deoxyguanosine sites in complementary DNA strands of Watson-Crick duplexes.
