Internal 32P-labeling of L-deoxyoligonucleotides.
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.
Frauendorf
Christian
C
NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, D-10589 Berlin, Germany.
Hausch
Felix
F
Röhl
Ingo
I
Lichte
Andrea
A
Vonhoff
Stefan
S
Klussmann
Sven
S
D017122:Bacteriophage T4 / Q000201:enzymology; D007553:Isotope Labeling / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D013379:Substrate Specificity
D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D011116:Polynucleotide 5'-Hydroxyl-Kinase