Ligation of the hairpin ribozyme in cis induced by freezing and dehydration

Abstract

Although reducing the temperature slows most chemical reactions, freezing can stimulate some reactions by mechanisms that are only partially understood. Here we show that freezing stimulates the self-ligation (circularization) of linear forms of the hairpin ribozyme (HPR) containing 2′,3′-cyclic phosphate and 5′-OH termini. Divalent metal ions (M²⁺) are not required, but monovalent cations and anions at millimolar concentrations can have various effects on this reaction depending on the specific ion. Under optimal conditions, the observed rate of M²⁺-independent self-ligation reaches a peak (0.04 min⁻¹) at −10°C with a yield of −60% after 1 h. In contrast, no ligation occurs either at above 0°C or in solutions that remain unfrozen when supercooled to subzero temperatures. Under freezing conditions, the cleavage–ligation equilibrium strongly favors ligation. Besides freezing, evaporation of the aqueous solvent as well as the presence of ethanol at levels of 40% or above can also induce M²⁺-independent HPR ligation at 25°C. We argue that partial RNA dehydration, which is a common feature of freezing, evaporation, and the presence of ethanol, is a key factor supporting HPR ligation activity at both above- and below-freezing temperatures. In the context of the RNA world hypothesis, freezing-induced ligation is an attractive mechanism by which complex RNAs could have evolved under conditions in which RNA was relatively protected against degradation.

Keywords

• hairpin ribozyme
• ligation
• freezing
• dehydration
• ethanol
• RNA world