The primary aim of this research is to develop a general method for covalently linking amine-containing molecules to the 5'-terminal phosphate of DNA or RNA without affecting the ability of the nucleic acid to hybridize with its complement. We will activate the unprotected nucleic acid in aqueous solution at pH 6 and room temperature with a water-soluble carbodiimide in an imidazole buffer. The 5' -phosphorimidazolide formed in this way will be isolated and then treated with a concentrated solution of the amine. This will lead to the formation of a stable covalent phosphoramidate adduct. In other experiments the 5' -terminal phosphate of DNA or RNA will be converted to a phosphoramidate without the isolation of an intermediate. The condensation reaction will be carried out in a 1-MeImidazole buffer. The very reactive phosphorimidazolium ion that is formed will immediately react with amines to give the corresponding phosphoramidates. These reactions will be used to prepare non-radioactive DNA probes, to prepare reagents that break DNA in a sequence-specific fashion, and to develop a non-enzymatic ligation reaction for DNA.
| Chu, B C; Orgel, L E (1994) Postsynthesis functionalization of oligonucleotides. Methods Mol Biol 26:145-65 |
| Chu, B C; Orgel, L E (1991) Binding of hairpin and dumbbell DNA to transcription factors. Nucleic Acids Res 19:6958 |
| Chu, B C; Orgel, L E (1990) Optimization of the efficiency of cross-linking PtII oligonucleotide phosphorothioate complexes to complementary oligonucleotides. Nucleic Acids Res 18:5163-71 |
| Chu, B C; Orgel, L E (1990) A simple procedure for cross-linking complementary oligonucleotides. DNA Cell Biol 9:71-6 |
