A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme

Abstract

The RNA world hypothesis suggests that chemical networks consisting of functional RNA molecules could have constructed a primitive life-like system leading a first living system. The chemical evolution scenario of RNA molecules should be consistent with the Hadean Earth environment. We have demonstrated the importance of the environment at both high temperature and high pressure, using different types of hydrothermal flow reactor systems and high-pressure equipment. In the present study, we have attempted to develop an alternative easy-to-implement method for high-pressure measurements and demonstrate that the system is applicable as an efficient research tool for high-pressure experiments at pressures up to 30 MPa. We demonstrate the usefulness of the system by detecting the high-pressure influence for the self-cleavage of avocado hammerhead ribozyme (ASBVd(−):HHR) at 45–65 °C. A kinetic analysis of the high-pressure behavior of ASBVd(−):HHR shows that the ribozyme is active at 30 MPa and its activity is sensitive to pressures between 0.1–30 MPa. The surprising finding that such a short ribozyme is effective for self-cleavage at a high pressure suggests the importance of pressure as a factor for selection of adaptable RNA molecules towards an RNA-based life-like system in the Hadean Earth environment deep in the ocean.

Life on Minerals: Binding Behaviors of Oligonucleotides on Zirconium Silicate and Its Inhibitory Activity for the Self-Cleavage of Hammerhead Ribozyme

“…Viroids are parasitic bodies that are sometimes considered as living fossils in relation to the RNA world. Recently, we showed a compensation effect of relatively high temperature and very high pressure, i.e., conditions that may be considered a realistic simulation of the deep ocean environment, for the self-cleavage of viroids and ribozymes [9,10]. This suggests that the submarine hydrothermal vent system in the deep ocean at relatively high temperature and high pressure would have been suitable for the chemical evolution of functional RNA molecules.…”

“…ASBVd(−):HHR (79 nucleotide units in length) was cleaved into two components of 51nt and 28nt to optimize the HPLC method for the separation of the ribozyme (79nt) and its two cleavage components (28nt and 51nt) [10,26]. HPLC analysis was carried out using an Infinity II chromatograph (Agilent, Santa Clara, CA, USA) with an anion-exchange column (diameter 4.6 mm and length 75 mm, DNA-NPR, TOSOH, Japan) at flow rate of 0.75 mL/min using a gradient of 0.375-0.975 M NaCl at pH 9.0 with 7.5 M urea and 0.02 M Tris buffer.…”

“…We investigated the self-cleavage reactions of a hammerhead ribozyme ASBVd(−): HHR and viroid ASBVd(−) at high pressure and temperatures [9,10,26]. So far, there have been few studies on the chemical behavior and the self-cleavage reactions of such a ribozyme and viroid on binding with minerals.…”

“…ASBVd45 binds with montmorillonite in large amounts in the presence of MgCl 2 and does not bind in the absence of MgCl 2 . According to our previous studies, chloride was selected as a counter ion for the magnesium ion [10,26]. In addition, dna-ASBVd45 binds onto zirconium silicate in the presence of MgCl 2 , but the binding significantly decreases in the absence of MgCl 2 .…”