The Synthesis of Lariat‐RNAs and their Conformational Analysis by NMR Spectroscopy: The Study of their Unique Self‐Cleavage Reaction Modelling Some Catalytic RNAs (Ribozymes)

The synthesis of milligram quantities of lariat RNAs 1, 2, 5-7 and their cyclic analogs 3 and 4, modelling the lariat formed at the penultimate step of ligation of Group II and Nuclear mRNA introns in the Splicing 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 the lariat-RNAs have been found to undergo site specific self-cleavage reaction to give an acyclic branched-RNA with 2′,3′-cyclic phosphate and a 5′-hydroxyl termini, which is reminiscent of the products formed in some catalytic RNAs. These lariat-RNAs are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k =1 min−1 at 37°C), and their rates of the self-cleavage is also much slower (k = 0.25×10−4 min−1 for lariat hexamer 5, and 0.16×10−3 min−1 for lariat heptamer 6 at 22°C). We have shown that the trinucleotidyl loop in the tetrameric 1 and pentameric 2 lariat-RNAs is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric 5 or heptameric 6 lariat-RNA does indeed have the required local and global conformation promoting the self-cleavage while the simple 2′5′ or 3′5′-linked cyclic RNAs, 3 and 4, respectively, are completely stable and their structures are considerably different from the self-cleaving lariat-RNAs such as 5 or 6. The unique 3′-ethylphosphate function at the branch-point in 7, mimicking the 3′-tail of the lariat-hexamer 5, is the key structural feature that orchestrates its self-cleavage reaction (k = 0.15×10−4 min−1 at 19°C) compared to the stable 2′5′-linked cyclic RNA 3. The detailed conformational features of the self-cleaving lariat-RNAs 5, 6 and 7 by 500 MHz NMR spectroscopy and molecular dynamics simulations in the aqueous environment has been reviewed. A comparative study of the temperature dependence of the N°S equilibrium for the lariat tetramer 7 and the 2′5′-linked cyclic tetramer 3 shows that the A1 residue in 7 is in 92% S-type conformation at 20°C, whereas it is only in 55% S in 3 with a 3′-hydroxyl group. This displacement of the N°S pseudorotational equilibrium toward the S geometry is due to the enhanced gauche effect of the 3′-OPO3Et− group at the branch-point adenosine in 7 compared to 3′-OH group in 3. This 3′-OPO3Et− group promoted stabilisation of the S geometry at the branch-point by ΔH = 4 kcal.mol−1 in 7 is contributing to the conformational driving force promoting its unique self-cleavage reaction. The comparison of ΔH and ΔS of the N°S pseudorotational equilibria in 7 and 3 clearly shows the remarkable effect of the 3′-ethylphosphate group in 7 in being able to dictate the conformational changes from the sugar moiety of the branch-point adenosine to the entire molecule (conformational transmission). Thus the S conformation in A1, U2 and C6 sugar moieties is clearly thermodynamically more stabilised while it is considerably destabilised in G3 owing to the 3′-ethylphosphate 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 A1 sugar in 7 is comparable to the enthalpy and entropy of transition between the A- and B-form of the lariat hexamer 5. This self-cleaving tetrameric lariat-RNA 7 is the smallest RNA molecule 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.