B to Z transactions in synthetics polynucletides

Abstract

The double helical structure of DNA proposed by Watson & Crick, which paved the way for the development of Molecular Biology, did not emphasize the importance of the sequence dependence of conformation. As a result, the DNA molecule was considered to be relatively monotonous in structural terms. However, the last decade has witnessed dramatic changes in earlier concepts through both theoretical studies on polynucleotide conformation and X?ray crystallographic studies of oligonucleotides.

The work reported in this thesis was undertaken as part of the ongoing programme of the laboratory aimed at understanding the role of specific DNA sequences in stabilizing the left?handed double helical structures and their biological implications. The availability of the structure of left?handed Z?DNA at atomic resolution prompted us to look in detail into the factors controlling structural transitions involving changes in handedness. Consequently, this study was concentrated on the mechanism of structural transitions, with special reference to B ? Z transitions taking polynucleotides and oligonucleotides as model systems.

The following aspects of the B ? Z transitions were investigated by the author:

Temperature?dependent B ? Z transitions in poly(dG?dC) under conditions of low dielectric constant.

Kinetics of the B ? Z transitions in poly(dG?dC) and poly(dG?m?dC) under different salt and solvent conditions.

Low salt B ? Z transitions in poly(dG?m?dC) and the effect of ions and temperature on such transitions.

Ant synergistic effects of phosphate ions and ethidium bromide on the three polynucleotides — poly(dG?dC), poly(dG?m?dC) and brominated poly(dG?dC) — in the presence of agents which stabilize Z?DNA.

Investigation of the role of base sequences in B ? Z transitions through studies on two chemically synthesized decanucleotides, d(CGTACGTACG) and d(CGTGCGCACG).

Chapter 1 of the thesis presents in brief the major advances that have taken place in the field of DNA structure prior to the discovery of Z?DNA, and provides a comprehensive description of Z?DNA. Also given in Chapter 1 is a survey of B ? Z transitions in various systems that are not directly related but relevant to the present study.

Chapter 2 describes the general methodologies employed during the course of this work. These include the experimental procedures involved in various spectroscopic, chromatographic and electrophoretic techniques which were used to carry out the present investigations.

Chapter 3 presents the effect of temperature on the B ? Z transitions of poly(dG?dC) under conditions of low dielectric constant. It was observed for the first time that the B ? Z transition is non?isothermal. The left?handed Z forms stabilized by 60% (v/v) aqueous ethanol underwent a reversible cooperative transition to the B form upon increasing the temperature. The midpoint of the temperature?induced transition was lowered in the presence of additional monovalent ions. An analysis of the temperature?dependent equilibrium constants for the Z ? B transitions yielded a positive enthalpy change.

Chapter 4 discusses the kinetics of B ? Z transitions in poly(dG?dC) and poly(dG?m?dC). The rate constants of the B ? Z transitions in these polymers in the presence of various Z?inducing agents were measured and found to be of the order of 10?³?sec?¹. The transitions induced by NaCl, MgCl? and hexamine cobalt(III) were found to be intramolecular in nature, while those in the presence of ethanol were found to depend on polymer concentration, indicating involvement of an intermolecular process.

Chapter 5 deals with the effect of phosphate ions on HCC?induced B ? Z transitions of poly(dG?dC). It was found that phosphate ions present in the medium had an inhibitory effect on the B ? Z transition brought about by HCC. Similarly, the destabilizing effect of ethidium bromide on the Z form of poly(dG?dC), poly(dG?m?dC) and brominated poly(dG?dC) was investigated.

The initial observations on the B ? Z transitions showed that an increase in the salt concentration of the medium was one of the factors which brought about such transitions. It was also observed that in poly(dG?m?dC) a B ? Z transition took place on decreasing the monovalent ion concentration as well. These results are outlined in Chapter 6.

The effect of various cations and anions known to have different solvent?structure making and breaking properties on the low salt B ? Z transition was studied in detail. Mg²?, which is known to stabilize the Z(II) conformation, was found to lock the low salt Z?conformation. The possibility of multivalent ion contamination being responsible for the Z?form was ruled out by performing experiments with extensively purified samples.

Chapter 7 summarizes studies on the effect of temperature on low and high salt B ? Z transitions of poly(dG?m?dC). It was observed that higher temperatures stabilized the Z conformation, regardless of the concentration of monovalent ions. The interplay between temperature and salt concentration was exploited to construct a phase diagram. It was found that extremely small changes in temperature or salt concentration were sufficient to cause a B ? Z transition in poly(dG?m?dC). Based on this temperature dependence and the existence of Z?conformation in low salt solutions, a mechanism for B ? Z transitions with special reference to methylation of cytosine was proposed.

Several deoxyoligomers with alternating purine?pyrimidine sequences (having both A?T and G?C base pairs) have been shown to adopt Z structure in the solid state. However, in solution, the Z form is favoured only when some of the bases in these sequences are covalently modified. The exact requirement of sequence or chain length for an oligonucleotide containing all four bases for a B ? Z transition to occur is not known. With this in view, two decanucleotides d(CGTACGTACG) and d(CGTGCGCACG) were synthesized by the phosphoramidite method and spectroscopic studies were carried out. Chapter 8 presents the details of synthesis and structural studies on these two decamers. Melting studies showed that these two decamers existed in the duplex form at room temperature. NaCl and HCC titration indicated that both sequences did not undergo B ? Z transition even at concentrations one order higher than that required for poly(dG?dC). On comparison with the crystal structures of Z?form, it appears that a continuous stretch of CG residues is necessary for an easy B ? Z transition to take place in solution even for a GC rich sequence.

Papers Published/Under Preparation:

B ? Z transitions in DNA and its biological implications — Proceedings of the INDO?FRG Seminar… BARC, Bombay, 1983.

Temperature dependent Z ? B transition in poly(dG?dC) — Curr. Sci., 1983.

A novel structural transition in poly(dG?m?dC): Z ? Z — FEBS Lett., 1985.

B ? Z transitions and their biological implications — J. Scientific Industrial Res., 1986 (in press).

…etc.