Conjugation of enzymes on RNA probes through Cu(I) catalyzed alkyne-azide cycloaddition

Northern and Southern blots are the most commonly used techniques for the confirmation of presence and expression of target genes. Molecular tools available for this purpose include radioisotope-, enzyme- and hapten-labeled nucleic acid probes. In particular, the use of enzyme-labeled probes are easy and safe, and do not require bound/free processes after hybridization associated with an antibody-based detection system. However, there are few approaches that enable the post-transcriptional modification of RNA with enzymes or proteins. In this study, we applied the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) reaction to the labeling of an RNA strand with enzymes. The C-5 position of UTP was modified with an alkyne group and alkyne-bearing RNA was prepared by in vitro transcription using T7 RNA polymerase. Surface amino groups of bacterial alkaline phosphatase (BAP) were randomly derivatized with azide groups at different modification ratios. The CuAAC reaction occurred selectively between the alkyne-modified RNA and the azide-modified enzyme. The RNA probe conjugated with BAP using this technique could detect a specific RNA by dot blot northern hybridization.