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

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions

“…The native gel electrophoretic characterization showed that <hairpin-ΔU> alone behaved as a monomer, but <A> alone behaved as both a monomer and dimer ( Figure 3 ). However, the combination of <A> and <hairpin-ΔU> showed the major formation of a hetero-dimer ( Figure 3 ), which could be derived from the kissing-loop interaction-induced rearrangement, consistent with our previous results [ 13 ]. Using these positions as controls, we found that the combinations of R3Ci<A>Cy5 and HHi<U>, R3Ci<A>Cy5 and <hairpin-ΔU>, and <A> and HHi<U> also formed dimers ( Figure 3 ).…”

“…Our previous study showed that the truncation of the R3C ligase ribozyme resulted in a marked decrease in ligation activity ( Figure 1 ) [ 11 ]. One such truncated mutant with a seven-membered single-stranded nucleotide (5′-AAUAACA-3′) is <A> (49-mer) ( Figure 1 B) [ 12 , 13 ]. <hairpin-ΔU> (22-mer) is a mutant of the R3C ligase ribozyme with a complementary seven-membered single-stranded nucleotide (5′-UGUUAUU-3′), but with no “grip” and no substrate-binding site (SBS) ( Figure 1 B) [ 12 , 13 ].…”

“…One such truncated mutant with a seven-membered single-stranded nucleotide (5′-AAUAACA-3′) is <A> (49-mer) ( Figure 1 B) [ 12 , 13 ]. <hairpin-ΔU> (22-mer) is a mutant of the R3C ligase ribozyme with a complementary seven-membered single-stranded nucleotide (5′-UGUUAUU-3′), but with no “grip” and no substrate-binding site (SBS) ( Figure 1 B) [ 12 , 13 ]. R3Ci<A>Cy5 (56-mer) is a 5′-Cy5-labeled mutant with extra nucleotides at the 5′-terminus of <A> ( Figure 2 ).…”

“…The reaction was performed according to the method of Rogers and Joyce with a slight modification [ 10 , 11 , 12 , 13 ]. The combination of 5 µM RNA(s) shown in Figure 4 dissolved in a solution containing 50 mM Tris-HCl (pH 8.5) and 15 mM MgCl 2 was first heated to 37 °C for 5 min and then cooled to 4 °C.…”

“…Furthermore, in the process of elucidating the “back to life” mechanism of inactive shortened R3C ribozymes ( Figure 1 ) [ 10 , 11 ], we discovered that the introduction of complementary seven-membered kissing loops revives their ligation activity [ 10 , 11 ]. This phenomenon suggests that a kissing complex formation induces a functional trans -rearrangement of two nonfunctional shortened R3C ribozymes [ 12 , 13 ]. The acquisition of a function through a kissing-loop interaction-induced rearrangement is not limited to the case of R3C ribozymes.…”