REMARKABLE CONFORMATIONAL CHANGE PROMOTED BY 3'-ETHYLPHOSPHATE AT THEBRANCH-POINT OF A TETRAMERIC LARIAT-RNA DICTATES ITS SELF-CLEAVAGE REACTION MODELING SOME CATALYTIC RNAS (RIBOZYMES)

Small synthetic lariat RNAs have been found to undergo site specific s elf-cleavage to give an acyclic branched-RNA with 2'9'-cyclic phosphat e and a 5'-hydroxyl termini, which is reminiscent of the products form ed in some catalytic RNAs. These lariat-RNAs are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k = similar t o 1 min(-1) at 37 degrees C), and their rate of the self-cleavage is a lso much slower (k = 0.25 x 10(4) min(-1) for lariat hexamer 18, and 0 .16 x 10(-3) min(-1) for lariat heptamer 19 at 22 degrees C). We have shown that the trinucleotidyl loop in the tetrameric and pentameric la riat-RNAs (ref. 10) is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric or heptameric lariat-RNA (re f. 10-13) does indeed have the required local and global conformation promoting the self-cleavage. It has been also shown that simple 2'-->5 ' or 3'-->5'-linked cyclic RNAs, 16 and 17, respectively, ore complete ly stable and their structures are considerably different from the sel f-cleaving lariat-RNAs such as 18 or 19. In our search to explore the optimal structural requirement for the self-cleavage reaction of RNA, we have now synthesized 14 in which the branch-point adenosine has a 2 '-->5'-linked tetranucleotidyl loop and a 3'-ethylphosphate moiety mim icking the 3'-tail of the lariat-hexamer 18. We here report that the u nique 3'-ethylphosphate function at the branch-point in 14 is the key structural feature that orchestrates its self-cleavage reaction (k = 0 .15 x 10(4) min(-1) at 19 degrees C) compared to the stable 2'-->5'-li nked cyclic RNA id (see Fig. 1). We also report the detailed conformat ional features of the self-cleaving tetrameric lariat-RNA 14 by 500 MH z NMR spectroscopy and Molecular dynamics simulations in the aqueous e nvironment. A comparative study of the temperature dependence of the N reversible arrow S equilibrium for the lariat-tetramer 14 and the 2'- ->5'-linked cyclic tetramer 16 shows that the A(1) residue in 14 is in 92% S-type conformation at 20 degrees C, wheras it is only in 55% S i n 16 with a 3'-hydroxyl group. This displacement of the N reversible a rrow S pseudorotational equilibrium toward the S geometry is due to th e enhanced gauche effect of the 3'-OPO(3)Et- group at the branch-point adenosine in 14 compared to 3'-OH group in Id, This 3'-OPO(3)Et-group promoted stabilization of the S geometry al the branch-point by Delta H approximate to 4 kcal.mol(-1) in 14 is the conformational driving f orce promoting its unique self-cleavage reaction. The comparison of De lta H degrees and Delta S degrees of the N reversible arrow S pseudoro tational equilibria in 14 and 16 (see Table 5) clearly shows the remar kable effect of the 3'-ethylphosphate group in 14 in being able to dic tate the conformational changes from the sugar moiety of the branch-po int adenosine to the entire molecule (conformational transmission. Thu s the S conformation in A(1) U-2 and C-6 sugar moieties is clearly the rmodynamically more stabilized while it is considerably destabilized i n G(3) owing to the 3'-ethylphosphate group in 14 compared to 16. It i s interesting to note that the magnitude of enthalpy and entropy for t he North to South transition of the A(1) sugar in 14 is comparable to the enthalpy and entropy of transition between the A- and B-form of th e lariat hexamer 18 (ref. 12) This self-cleaving tetrameric lariat-RNA 14 is the smallest RNA molecule hitherto known to undergo the self-cl eavage reaction and hence it is the simplest model of the active cleav age site of the natural self-cleaving catalytic RNA.