Vitamin C for peptide ligation: Phosphine and thiol additives maintain reducing environments and increase the reactivities of peptide thioesters in native chemical ligation. Thiol additives such as thiophenol also act as radical scavengers that inhibit phosphine‐induced desulfurization of cysteine. This role can be assumed by the odourless, nontoxic, highly water‐soluble and inexpensive ascorbate, which can replace the usually added thiols.
Two peptides on display: The self‐assembly of DNA complexes enables the bivalent presentation of phosphopeptides. Flexibility and distance in the ligand arrangement can be adjusted through the choice of appropriate DNA templates. Spatial screening of the tandem SH2 domain of Syk kinase with these probes (see picture) indicated the accessible arrangements of the two homologous binding pockets and the flexibility of the connecting protein linker.
The self-assembly of nanosized DNA templates--based on formation of duplex, triplex, quadruplex or even pentaplex structures--provides unique opportunities for the controlled presentation of appended functional units. Recently, researchers have recognized the potential of such DNA scaffolds to address questions in the life sciences. In this critical review the focus is on the exploration of proteins. It is shown how different scaffolds can be used to control localization, structure and bioactivity of proteins and protein ligands. Further examples demonstrate that DNA-based recognition can even be used to trigger the formation of protein targeted molecules. Potential and existing applications in protein detection, drug discovery, structural characterization of protein targets as well as in the design of nucleic acid responsive pharmacophores are discussed (107 references).
Peptide-oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime-, thioether-, or disulfide-linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self-assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu-protected cysteine was attached via an aminopropargyl linker to the C5-position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5-6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide-oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes.
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