Internal P-32-labeling of L-deoxyoligonucleotides - art. no. e34

摘要

A general two step procedure for the internal labeling of L-deoxyoligonucleotides, Spiegelmers, has been developed. Through radioactive labeling oligonucleotides can easily be detected and monitored in biological samples. T4 polynucleotide kinase is shown to efficiently phosphorylate strands of L-nucleic acids which allows the labeling with phosphorous isotopes such as P-32. In order to protect the terminal phosphate label against unspecific phosphatases, one of two fragments of a Spiegelmer is enzymatically phosphorylated with [gamma-P-32] ATP. In a second step we used a template-directed chemical ligation reaction in order to attach the labeled oligonucleotide to the other fragment to yield the full-length Spiegelmer with an internal [P-32] phosphodiester bond. It has been shown that the functionality of a chemically ligated Spiegelmer is still retained.