Site-Specific Incorporation of Fluorescent Isomorphic G Surrogate into Hairpin RNA to Monitor RIPs Depurination in Real-time
- Author(s): Cong, Deyuan
- Advisor(s): Tor, Yitzhak
- et al.
Ribosome-inactivating proteins (RIPs), a family of highly cytotoxic proteins, inhibit protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop (SRL) of eukaryotic ribosomal RNA. Enzymatic activity of RIPs become a fundamental target for preventing the toxicity of RIPs. The fluorescent method is considered to be a potentially effective approach to monitor the depurination reaction in real-time. Herein, we describe an enzyme-mediated approach in which specific G residues besides the active site are surgically replaced with fluorescent analog tzG and thG by transcription, phosphorylation and ligation. By strategically modifying the key position, the kinetic profile in depurination is obtained. In the contrast with traditional 32P labeling results, good agreements in the apparent rate of kinetics prove the singly modified fluorescent strand is sensitive to microenvironmental change and could monitor the enzymatic reaction in real-time by steady-state fluorescence spectroscopy. The difference in active site between the tzG strand and thG strand indicates N7 position in purine ring plays an important role in most of the biochemical reactions in terms of stabilizing secondary structure.