Freely diffusing single hairpin ribozymes provide insights into the role of secondary structure and partially folded states in RNA folding.

Single-molecule fluorescence resonance energy transfer studies of freely diffusing hairpin ribozymes with different combinations of helical junction and loop elements reveal striking differences in their folding behavior. We examined a series of six different ribozymes consisting of two-, three- and four-way junction variants, as well as corresponding constructs with one of the two loops removed. Our results highlight the varying contributions of preformed secondary structure elements to tertiary folding of the hairpin ribozyme. Of the three helical junction variants studied, the four-way junction strongly favored folding to a docked conformation of the two loops, required for catalytic activity. Moreover, the four-way junction was uniquely able to fold to a similar compact structure even in the absence of specific loop-loop docking interactions. A key feature of the data is the observation of broadening/tailing in the fluorescence resonance energy transfer histogram peak for a single-loop mutant of the four-way junction at higher Mg(2+) concentrations, not observed for any of the other single-loop variants. This feature is consistent with interconversion between compact and extended structures, which we estimate takes place on the 100-micros timescale using a simple model for the peak shape. This unique ability of the four-way junction ribozyme to populate an undocked conformation with native-like structure (a quasi-docked state) likely contributes to its greater tertiary structure stability, with the quasi-docked state acting as an intermediate and facilitating the subsequent formation of the specific hydrogen bonding network during docking of the two loops. The inability of two- and three-way junction ribozymes to fully populate a docked conformation reveals the importance of correct helical junction geometry as well as loop elements for effective ribozyme folding.