ABSTRACT
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).
Subject(s)
Biology/methods , DNA/metabolism , Proteins/metabolism , Animals , Biosensing Techniques , DNA/chemistry , Humans , Ligands , Nucleic Acid Conformation , Proteins/chemistryABSTRACT
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.
Subject(s)
Oligonucleotides/chemical synthesis , Peptides/chemical synthesis , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotides/chemistry , Peptides/chemistryABSTRACT
The search for novel, generally applicable and highly efficient delivery tools is a major activity in the biotechnology arena. Using highly optimized microwave based solid-phase chemistry a series of fluorescein-labelled cationic peptoid conjugates were synthesized within 24h and cellular uptake into HeLa, L929 and K562 cells examined via flow cytometry. As expected, analysis revealed that longer oligomers achieved greater cellular penetration (7e (9 mer)>7d (7 mer)>7c (5 mer)>7b (3 mer)>7a (1 mer)) with the nonamer 7e proving to be a remarkable vehicle for all the cell lines, showing excellent penetrability into K562 and L929 cells and extraordinary cell delivery into HeLa cells. Confocal microscopy showed that the hybrid peptoid-nuclear localizing sequence (PKKKRKV from the simian virus 40 large T antigen) resulted in very high levels of nuclei delivery after 3h, opening up a range of applications such as nuclei staining of living cells with non-DNA-intercalating fluorescent probes.
