Site-specific crosslinking of a p19 viral suppressor of RNA silencing protein and its RNA targets using an expanded genetic code.

The p19 viral suppressor of RNA silencing protein binding to small RNAs such as small interfering RNAs (siRNAs). has useful applications in biotechnology due to its high affinity for Also, its applications for the study and modulation of microRNAs are actively expanding. Here we demonstrate the successful site-specific incorporation of a photoactivatable unnatural amino acid, p-azido-L-phenylalanine (AzF), for cross-linking to RNA substrates into the p19 sequence. Incorporation of AzF was performed at three positions in the protein near the RNA binding site: K67, R115, and T111. Incorporation of AzF at position T111 of p19 did not affect the binding affinity of p19 for siRNAs and also showed nanomolar affinity for human microRNA miR-122. The affinity was less favorable with AzF incorporation at two other positions, suggesting the sensitivity of placement of the unnatural amino acid. Exposure of the T111AzF in complex with either siRNA or miRNA to ultraviolet light resulted in cross-linking of the protein with the RNA, but no cross-linking could be detected with the wild-type protein. Our results demonstrate that p19-T111AzF can be used for detection of small RNAs, including human miR-122, with high sensitivity and to irreversibly sequester these RNAs through covalent photo-cross-linking.