3'-end labeling of RNA with recombinant yeast poly(A) polymerase

Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.

Published in:
Nucleic Acids Res, 21, 12, 2917-20
Department of Cell Biology, Biozentrum of the University of Basel, Switzerland.

 Record created 2007-11-20, last modified 2019-12-05

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