Cobalt(III)hexaammine-dependent photocrosslinks in the hairpin ribozyme

• Cobalt(III)hexaammine mediates formation of photocrosslinks in the hairpin ribozyme.
• The yield of crosslinks is pH-dependent.
• The two main photocrosslinks formed in this fashion are catalytically active.
• The oxidation potentials of the bases involved affect the yield of the crosslinks.
• The crosslinks require stacking and close interaction of the two donor bases.
• This tool has proven useful for analysis of the hairpin ribozyme folded structure.

We have utilized the hairpin ribozyme, an RNA enzyme whose structure has been solved by high-resolution methods, to develop a new tool for mapping nucleobase–stacking interactions and potential metal-binding sites in RNA molecules. This tool involves the photoactivation of a specifically bound cobalt(III)hexaammine molecule at wavelengths corresponding to excitation of the metal ion complex only; no base excitation is involved. The photoexcitation initiates a process which strongly promotes the formation of a novel covalent bond or crosslink between one base (termed the “first base”), which is close in space to the excited cobalt(III)hexaammine complex, and another base upon which the first base is closely stacked. These crosslinked species can be isolated and sequenced; their activities can be analyzed to ensure that the crosslinked structures represent an active conformation of the molecule. We have shown that, as in electron transfer in DNA, several criteria must be met to result in the successful formation of these crosslinks. These include the appropriate oxidation potential of the first donor base, the stacking and close interaction of the two donor bases involved in the crosslink, and the binding of a specific cobalt(III)hexaammine molecule to the first donor base. Additionally, we have determined that this crosslinking is pH-sensitive, although the cause of this sensitivity remains unknown. This tool has proven useful in the past for the analysis of the hairpin ribozyme folded structure, and has been applied to identify potential metal-binding sites on the hairpin and extended hammerhead ribozymes.

Cobalt(III)hexaammine, Co(NH3)63 +, mediates the formation of photocrosslinks in the hairpin ribozyme, a small RNA enzyme. These photocrosslinked species retain functional activity. The yields of these species depend on several factors, including the pH, oxidation potential of the first donor base and the binding of a specific Co(NH3)63 +.Figure optionsDownload as PowerPoint slide