Simple methods for the 3' biotinylation of RNA.
Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.
Bodo
B
Moritz
Elmar
E
Wahle
D000255:Adenosine Triphosphate / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D029702:Schizosaccharomyces pombe Proteins; D012313:RNA; D001710:Biotin; D000255:Adenosine Triphosphate; D019809:Streptavidin; D009713:Nucleotidyltransferases; C520554:Cid1 protein, S pombe; D011062:Polynucleotide Adenylyltransferase; D004259:DNA-Directed DNA Polymerase
3′-end modification; RNA tagging; biotin