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

Publisher Website
Google Scholar
Abstract
Two commonly used methods to end-label RNA-molecules are 5′end labeling by potynucleotide kinase and 3′-end labeling wlth 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) poiymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.