Behavior of DNA under hydrothermal conditions with MgCl₂ additive using an in situ UV–vis spectrophotometer

Abstract

The capillary flow hydrothermal reactor system for the UV–vis spectrophotometric detection system (CHUS) has been applied to inspect the behavior of DNA at temperatures up to 300 °C. Double-stranded DNA (dsDNA) was monitored with and without ethidium bromide in the presence of MgCl₂. The ratio (A_+DNA/A_−DNA) of the absorbance of EB with DNA (A_+DNA) to that without DNA (A_−DNA) increased steeply over 1 at temperatures around 100 °C, which was not observed in the absence of MgCl₂. This was found to be due to the decrease of solubility of DNA in hot water, where dsDNA is converted to single-stranded DNA and become insoluble. At temperatures over 175 °C, DNA becomes soluble again because of the degradation of long insoluble DNA polymers to short soluble DNA oligomers. This study points out the importance of solubility of DNA for life at extremely high temperatures as well as the stability of DNA.

Introduction

Hydrothermal reactions are becoming important in both fundamental and practical areas. Nowadays, it is realized that hydrothermal environments are indispensable for some special biochemical reactions. First, the hypothesis that life could have been originated under hydrothermal environments (the hydrothermal origin of life hypothesis) has been proposed. The hydrothermal origin of life hypothesis is supported by the phylogenetic analysis of the present living-organisms and the simulation experiments under submarine vent conditions on the earth [1], [2], [3], [4], [5], [6]. The last common ancestor (LCA) of the present organisms is considered as a hyperthermophilic organism [1], [2], [3]. In addition, geological records suggest that the primitive Earth environments resembled the modern hyperthermophilic biotopes when oldest organisms were present [7], [8], [9], [10], [11]. However, the nature of LCA is not yet commonly accepted [12], [13], [14], [15]. Furthermore, the hydrothermal origin of life hypothesis seems to be inconsistent with the RNA world hypothesis [16], [17], [18], [19], [20]. That is to say, it is considered that nucleotides are labile under redox constrained hydrothermal conditions [12], [21], [22], [23], [24], [25], [26] although some minerals are capable to protect nucleotides and their precursors from their degradations [27]. Additionally hydrophobic interaction and hydrogen bonding within polynucleotides would not be effective to preserve biological information and catalytic functions at high temperatures [21], [28], [29]. Although the hydrothermal origin of life hypothesis has been still disputed, these speculations need to be experimentally verified.

Second example of hydrothermal reactions related with biological science can be found in the biochemistry of hyperthermophilic organisms. Presumably, DNA molecules are protected by several mechanisms in modern hyperthermophiles [30], such as specific proteins [31], polyamines [32], [33], increase of G–C ratio [34]. However, the melting of naked dsDNA molecules normally proceeds at temperatures below 100 °C [35], [36]. Thus, it would be essential to evaluate how tertiary structures and covalent bonding within DNA molecules could be stabilized in thermophiles at high temperatures near and over 100 °C.

Nevertheless, there has been no practical technique to evaluate the behaviors of biomolecules at extremely high temperatures. In addition, there are few studies on the solubility of biopolymers under hydrothermal conditions while biopolymers should be protected from their aggregation and precipitation even under hydrothermal conditions for preserving replication and catalytic functions in hyperthermophiles. These drawbacks concerning the origin of life and the biochemistry of thermophilic organisms have been experimentally verified by our new techniques for hydrothermal reactions [20], [25], [26], [28], [29], [37], such as by the capillary flow hydrothermal reactor system for the UV–vis spectrophotometric detection system (CHUS) to investigate hydrothermal reactions [38]. This technique was applied for evaluation of the behavior of DNA at 25–200 °C using the intercalation of ethidium bromide (EB) to dsDNA [29]. However, it was unexpected that the absorbance of EB at 110–135 °C was increased in the presence of phosphate or borate pH buffer. The scope of this unknown phenomenon has been continuously investigated.

In the present study, the observation of DNA behavior with MgCl₂ additive under the hydrothermal environments was attempted using CHUS to evaluate the stability and the tertiary structure of DNA at temperatures up to 300 °C since MgCl₂ potentially stabilizes dsDNA [36]. Based on the results, the importance of solubility of DNA molecules for life at extremely high temperatures is discussed.