Internal 32P-labeling of l-deoxyoligonucleotides

AUTOR(ES)

Frauendorf, Christian

FONTE

Oxford University Press

RESUMO

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 32P. In order to protect the terminal phosphate label against unspecific phosphatases, one of two fragments of a Spiegelmer is enzymatically phosphorylated with [γ-32P]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 [32P]phosphodiester bond. It has been shown that the functionality of a chemically ligated Spiegelmer is still retained.

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