Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, <A> and <hairpin-ΔU> the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in <hairpin-ΔU> but not in <A> despite the absence of a substrate-binding site in <hairpin-ΔU>. Loop-loop interactions of <A>- and <hairpin-ΔU>-variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.