Remarkable conformational change promoted by 3′-ethylphosphate at the branch-point of a tetrameric lariat-RNA dictates its self-cleavage reaction modelling some catalytic RNAs (ribozymes)

Abstract

Small synthetic lariat RNAs have been found to undergo site specific self-cleavage 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 rate of the self-cleavage is also much slower (k = 0.25 × 10 −4 min −1 for lariat hexamer 18, and 0.16 × 10 −3 min −1 for lariat heptamer 19 at 22 °C). We have shown that the trinucleotidyl loop in the tetrameric and pentameric lariat-RNAs (ref. 10) is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric or heptameric larita-RNA (ref. 10–13) does indeed have the required local and globla conformation promoting the self-cleavage. It has been also shown that simple 2′→5′ or 3′→5′-linked cyclic RNAs, 16 and 17, respectively, are completely stable and their structures are considerably different from the self-cleaving lariat-RNAs such as 18 and 19. In our search to explore the optimal 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 moeity mimicking the 3′-tail of the lariat-hexamer 18. We have report that the unique 4′-ethylphosphate function at the branch-point in 14 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 16 (see Fig. 1). We also report the detalied conformational features of the self-cleaving tetrameric lariat-RNA 14 by 500 MHz NMR spectroscopy and Molecular dynamics simlations in the aqueous environment. A comparative study of the temperature dependence of the N⇄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 °C, whereas it is only in 55% S in 16 with a 3t́-hydroxyl group. This displacement of the N⇄S pseudorototational equilibrium toward the S geometry is due to the enhanced gauche effect of the 3′-OPO 3Et − group at the branch-oint adenosine in 14 compared to 3′-OH group in 16. This 3′-OPO 3Et − group promoted stabilization of the S geometry at the branch-point by ΔH ≈ 4 kcal.mol −1 in 14 is the coformational driving force promoting its unique self-cleavage reaction. The comparison of ΔH° and ΔS° of the N⇄S pseudoorotational equilibria in 14 and 16 (see Table 5) clearly shows the remarkable effect of the 3′-ethylphosphate group in 14 in being able to dictate the conformational changes from the sugar moeity of the branch-point adenosine to the entire molecule (conformational transmission). Thus the S conformation in A 1, U 2 and C 6 sugar moieties is clearly thermodynamically more stabilized while it is considerably destabilized in G 3 owing to the 3 −-ethylphosphate group in 14 compared to 16. It is interesting to note that the magnitude of enthalpy and entropy for the North to South transition of the A 1 sugar in 14 is comparable to the enthalphy and entropy of transition between the A- and B-form of the lariat hexamer 18 ref. 12). This self-cleaving tetrameric lariat-RnA 14 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.