Internal 32P-labeling of L-deoxyoligonucleotides

Abstract

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

Figure 1

Comparative analysis of the T4 PNK with l-DNA 1–4 (the terminal nucleotides are indicated). 400 pmol of the respective l-DNA was incubated in kinase buffer with 40 U T4 PNK in the presence of [γ-³²P]ATP in a 20 µl reaction. Incubation was carried out at 37°C for 3 h. Products were visualized by autoradiography (20% denaturing polyacrylamide gel).

Figure 2

Autoradiogram of a 10% denaturing polyacrylamide gel of the phosphorylation reaction of 6 (lane a) and the internal radioactive labeling using chemical ligation of 30mer l-DNA 5 and phosphorylated 37mer l-DNA 6 on a 33mer template 7 (lane b) with BrCN. XC, xylene cyanol.