THE SYNTHESIS OF LARIAT-RNAS AND THEIR CONFORMATIONAL-ANALYSIS BY NMR-SPECTROSCOPY - THE STUDY OF THEIR UNIQUE SELF-CLEAVAGE REACTION MODELING SOME CATALYTIC RNAS (RIBOZYMES)

The synthesis of milligram quantities of lariat RNAs 1, 2, 5-7 and the ir cyclic analogs 3 and 4, modelling the lariat formed at the penultim ate step of ligation of Group II and Nuclear mRNA introns; in the Spli cing reaction, have been achieved for the first time. These syntheses are highlighted by unique employment of various orthogonal protecting groups using phosphotriester and phosphoramidite chemistry. Some of th e lariat-RNAs have been found to undergo site specific self-cleavage r eaction to give an acyclic branched-RNA with 2',3'-cyclic phosphate an d a 5'-hydroxyl termini, which is reminiscent of the products formed i n some catalytic RNAs. These lariat-RNAs are much smaller than the nat ural catalytic RNAs such as the hammerhead ribozyme (k = similar to 1 min(-1) at 37 degrees C), and their rates of the self-cleavage is also much slower (k = 0.25x10(-4) min(-1) for lariat hexamer 5, and 0.16x1 0(-3) min(-1) for lariat heptamer 6 at 22 degrees C). We have shown th at the trinucleotidyl loop in the tetrameric 1 and pentameric 2 lariat -RNAs is completely stable whereas the tetranucleotidyl or pentanucleo tidyl loop in the hexameric 5 or heptameric 6 lariat-RNA does indeed h ave the required local and global conformation promoting the self-clea vage while the simple 2'-->5' or 3'-->5'-linked cyclic RNAs, 3 and 4, respectively, are completely stable and their structures are considera bly different from the self-cleaving lariat-RNAs such as 5 or 6. The u nique 3'-ethylphosphate function at the branch-point in 7, mimicking t he 3'-tail of the lariat-hexamer 5, is the key structural feature that orchestrates its self-cleavage reaction (k = 0.15x10(-4) min(-1) at 1 9 degrees C) compared to the stable 2'-->5'-linked cyclic RNA 3. The d etailed conformational features of the self-cleaving lariat-RNAs 5, 6 and 7 by 500 MHz NMR spectroscopy and molecular dynamics simulations i n the aqueous environment has been reviewed. A comparative study of th e temperature dependence of the N double left right arrow S equilibriu m for the lariat tetramer 7 and the 2'-->5'-linked cyclic tetramer 3 s hows that the A(1) residue in 7 is in 92% S-type conformation at 20 de grees C, whereas it is only in 55% S in 3 with a 3'-hydroxyl group. Th is displacement of the N double left right arrow S pseudorotational eq uilibrium toward the S geometry is due to the enhanced gauche effect o f the 3'-OPO(3)Et(-) group at the branch-point adenosine in 7 compared to 3'-OH group in 3. This 3'-OPO(3)Et(-) group promoted stabilisation of the S geometry at the branch-point by Delta H approximate to 4 kca l.mol(-1) in 7 is contributing to the conformational driving force pro moting its unique self-cleavage reaction. The comparison of Delta H de grees and Delta S degrees of the N double left right arrow S pseudorot ational equilibria in 7 and 3 clearly shows the remarkable effect of t he 3'-ethylphosphate group in 7 in being able to dictate the conformat ional changes from the sugar moiety of the branch-point adenosine to t he entire molecule (conformational transmission). Thus the S conformat ion in A(1), U-2 and C-6 sugar moieties is clearly thermodynamically m ore stabilised while it is considerably destabilised in G(3) owing to the 3'-ethylyphosphate group in 7 compared to 3. It is interesting to note that the magnitude of enthalpy and entropy for the North to South transition of the A(1) sugar in 7 is comparable to the enthalpy and e ntropy of transition between the A- and B-form of the lariat hexamer 5 . This self-cleaving tetrameric lariat-RNA 7 is the smallest RNA molec ule hitherto known to undergo the self-cleavage reaction and hence it is the simplest model of the active cleavage site of the natural self- cleaving catalytic RNA.