Los reacomodos de la justicia y el nacimiento de la Constitución de 1991 (1987-1992)

The overall aim of this thesis is to provide the first chemical and physical studies examining the role of both pressure and temperature on chemical and physical stability of DNA and its constituents using predominantly NMR spectroscopy. This will fill in some of the large gaps in the understanding of the effects of high pressure on the chemical and physical stability of DNA, particularly the effects of both high temperature and high pressure. The outcomes of this research will be used to improve the current understanding of the potential role of high-pressure on the origin of self-replicating systems. It will also address some fundamental questions: Will high pressures have any effect on the chemical or physical stability of DNA? If so, will this be a positive or negative effect? And is this change in stability capable of sufficiently offsetting the destabilising effects, both chemical and physical, of high temperature on DNA and its constituent nucleobases?

Examination of the effects of high pressure on the chemical and physical stability of nucleic acids will be performed using DNA. The difficulty in removing RNases from equipment makes RNA extremely difficult to work with and will most likely lead to the loss of expensive RNA samples. DNA will be a suitable, more stable, alternative. The similarities of DNA to RNA (particularly the physical structure of

9 small duplexes) will allow the results obtained for DNA to be considered as acceptable analogues for RNA until more results are obtained.

High-pressure NMR spectroscopy will allow for the observation of the chemical composition of solutions as well as being able to observe physical behaviours as changes in chemical shift and intensity of NMR signals. This technique also has a great advantage in being able to observe the behaviours of each individual nucleotide in a DNA sequence rather than only observing the average behaviour as with other high-pressure spectroscopic techniques (such as UV-visible spectroscopy and circular dichroism spectroscopy).

This aim can be broken down into four major parts, which will each be independently studied. Detailed introductions are given within each of the research chapters, providing further information relevant to the enclosed studies.

1.4.1

Development of a High-Pressure System

To study DNA under high-pressure conditions, a specialised high-pressure system will be developed. This system will have to allow for the safe-handling and operation of a commercial high-pressure NMR cell as well as providing a mounting point for a high-pressure pump which will pressurise the whole system. A removable high-pressure reaction vessel will also be added to this system to extend its capabilities beyond the NMR spectrometer.

1.4.2

Chemical Stability of Individual DNA Bases

High-pressure NMR spectroscopy will be used to examine the effects of high pressure on the rate of hydrolysis of cytosine and cytidine (the least stable of the nucleobases and therefore a limiting factor) at 100°C. This will involve examining the rate of change of cytosine/cytidine concentrations as they decay, to uracil/uridine respectively (Figure 1.4). This will be repeated under multiple

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Figure 1.4: Equation for the Hydrolysis of Cytosine to Uracil.

pressure conditions to establish the reaction volume of cytosine/cytidine hydrolysis. A brief assessment of the rate of cytosine hydrolysis as a function of pH at both high and low pressures will also be performed.

1.4.3

Physical Stability of DNA Structures

High-pressure NMR spectroscopy will be used to examine the effects of high- pressure on the melting points of a self-complementary DNA hexamer and five self- complementary DNA dodecamers. This will first involve the use of NOESY and COSY 1_{H NMR techniques to assign the peaks of the} 1_{H NMR spectra for each of the}

DNA sequences. This will be followed by melting experiments which can be used to determine melting curves, and therefore melting points, for, in principle, each residue of each sequence under multiple pressure conditions.

The effect of high pressure on the physical stability of a DNA i-motif will also be examined. The melting point of the i-motif sequence will be determined using high- pressure NMR spectroscopy under different pressure and pH conditions.

These studies will develop an understanding of how the physical stability of the DNA sequences is affected by high-pressure.

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1.4.4

Chemical Stability of DNA

Next-generation sequencing technology will be used to determine the effect of high pressure and high temperature on the chemical stability of cytosine within both single-stranded and double-stranded bacteriophage DNA. Samples will be incubated under a variety of temperature and pressure conditions after which they will be sequenced to determine any cytosine hydrolysis (which will be seen as a cytosine to thymine transition) within the DNA sequence. With this ability to examine each cytosine in the sequence it will be possible to study the effect of high pressures on the chemical stability of each cytosine residue in the DNA sequence and to determine whether sequence position results in any differences in the chemical stability and the susceptibility of the chemical stability to pressure.