Studies on B. DNA fibre diffraction and ligand binding

Abstract

Studies on BDNA: Fibre Diffraction and Ligand Binding This thesis consists of theoretical and experimental work carried out by the author during the period August 1982 to October 1987. It is divided into two parts:

Part I: Xray fibre diffraction studies on BDNA Part II: Design, synthesis and physicochemical studies of structural analogues of the oligopeptide antibiotic distamycin, a BDNAspecific ligand

A thorough and systematic conformational analysis examining all possible helical structures for BDNA, using the flexible mononucleotide as the building block, was first initiated in our laboratory. As a result of these studies, several righthanded and lefthanded models were generated that were consistent with the then available Xray fibre data as well as IR dichroism data. At the time the author joined the laboratory, Xray fibre diffraction of DNA-specifically the Bform-had just been initiated. The objective was to supplement earlier modelbuilding work with firsthand, and ideally more accurate, fibre data for comparison with the various theoretical models. The first part of this thesis deals with such attempts and the results obtained. It was observed that for the lithium salt of calfthymus DNA, the diffraction pattern corresponding to the best “crystalline Bform” cannot be explained using regular helical structures. Additionally, structural transitions in the solid state, dependent on environmental conditions, were detected-highlighting the inherent flexibility of the structure earlier suggested by conformational calculations carried out in our laboratory. Chapter 1 presents a historical overview of studies on DNA structure, emphasizing the importance of Xray fibre diffraction and modelbuilding studies and their interdependence. After examining existing methods for fibre preparation, a new method for accurately controlling the salt content in DNA fibres-an essential factor for crystallinity and quality of Xray patterns-was developed in our laboratory. This method is described in Chapter 2, along with standardized procedures for obtaining highly reproducible crystalline BDNA patterns. Chapter 3 provides detailed Xray investigations using these methods. Appearance and disappearance of meridional reflections on the fourth, sixth, and tenth layer lines indicated nonuniformity and variability in structure. Sensitive and quantitative measurements of helical parameters from layerline spacings, using the precession method (applied to DNA fibres for the first time), confirmed such variations. Since these variations were observed even in the best Bpatterns and indicated a likely nonhelical nature, constructing accurate structural models consistent with all observed features became extremely difficult. It was concluded that the Xray data did not justify a detailed solution of the fine structure of BDNA. To explore gross structural features (e.g., handedness) and to attempt to “lock” the structure into one of its microheterogeneous states, the nonintercalative DNAbinding ligands netropsin (Nt) and distamycin (Dst) were studied. These naturally occurring oligopeptide antibiotics bind specifically to:

BDNA ATrich sequences the minor groove

Their structural complementarity to the BDNA minor groove makes them excellent models for designing structural probes. The author contributed to initiating this ligandbindingbased approach to DNA structure, forming the basis of Part II of this thesis. Chapter 4 introduces the distamycin-netropsin system and its relevance to DNA structure. An isohelical analysis of pyrrolamides was carried out to examine their compatibility with BDNA. A pyrrole-alanine repeat unit was identified as a viable design element for synthetic analogues. These results are described in Chapter 5. Chapter 6 describes synthetic procedures for preparing two pyrrole-alanine analogues of distamycin, PPA and PAP. Introduction of the saturated alanine moiety reduces the extended conjugation present in contiguous pyrrolamides (as in Dst). Detailed spectroscopic studies of PPA and PAP binding to DNA were conducted to examine the role of extended conjugation in Dst-DNA interactions. In Chapter 7, it is shown that loss of extended conjugation does not affect the specificity of binding. However, Chapter 8, the final chapter, demonstrates that conjugation contributes significantly to the stability of the ligand-DNA complex. Although ATspecificities of Nt, Dst, and their synthetic analogues are widely discussed, comparisons are typically qualitative. A more rigorous method for evaluating and comparing ATspecificity was developed based on results obtained in our laboratory. This procedure is presented in Appendix I. A new and conceptually simple derivation of Euler’s formula for combining rotational axes in crystallographic point groups is given in Appendix II.