A biophysical study of nucleic acid interactions with analogues and drugs

• work presented in this thesis concerns studies on the physicochemical nature of interactions between nucleic acids and small ligands. The outcome of such studies can yield insights at a molecular level into the physiological mechanisms of action of biologically active nucleic-acid binding molecules. The thesis work includes investigations of a number of such low molecular weight compounds designed for nucleic acid sequence probing or therapeutic use. The interactions have been characterised by means of various optical spectroscopic techniques - including linear dichroism, circular dichroism and fluorescence - as well as nuclear magnetic resonance spectroscopy.The fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) is known to adopt different DNA binding modes in regions containing consecutive AT base-pairs as compared to those consisting of long sequences of GC base-pairs. In mixed sequence DNA, DAPI exhibits a pronounced preference to bind in the minor groove of AT rich regions. To verify whether the variation in ligand mode of binding could be attributed to the exocyclic N2 group of guanine, guanines in the polynucleotide [poly(dG-dC)]2 were substituted for the 2-desamino analogue inosine, denoted I. Comparison of the spectral characteristics of DAPI in complex with either of the polymeric nucleic acid host structures revealed a clear difference in binding geometries; the spectroscopic properties of the IC complex closely resemble those of the AT complex, in which DAPI is inserted edgewisely along the minor groove. These results are rationalised in terms of steric hindrance and decreased electronegative attraction caused by the amino group protruding into the minor groove of B-type GC tracts.Studies of complexes between DNA and a series of cobalt porphyrins and their unmetallated analogues revealed contact energy transfer from the DNA bases and binding orientation angles nearly parallel to the planes of the DNA base-pairs, indicative of intercalation, for the non-metal porphyrins. The metalloporphyrins, on the other hand, are suggested to bind in a partially melted region of DNA. This hypothesis is supported by cleavage reactions in which a break in one of the DNA strands is induced by a single activation event on the cobalt porphyrins.The DNA interaction properties for a series of quinoxaline derivatives with a positively charged side chain were examined with respect to variation of the size of the molecular heterocyclic ring system. Derivatives with three, four, and five rings were included in the investigations. All but the tricyclic compound are found to bind in an intercalative mode irrespective of DNA base sequence. For the tricyclic derivative it is suggested that the binding involves a competitive equilibrium between intercalation preferred by the ring system, and minor groove binding favoured by the side chain.The second part of this thesis focuses on the interactions between nucleic acids and two compounds with the potential of being used in the novel therapeutic antigene/antisense strategies, either by means of its own action (PNA, peptide nucleic acid) or as an antigene enhancer (9-OH-B220).The binding of the biologically active quinoxaline derivative 9-OH-B220 to double and triple helices of synthetic DNA and RNA was characterised. The drug is found to adopt an intercalative binding geometry in all complexes except when the RNA triplex serves as a host structure. In the latter case, the spectroscopic properties are indicative of a binding of the drug chromophore in the wide and shallow minor groove of the RNA triplex polymer. The drug is also found to enhance the thermal stability of each nucleic acid structure, with the DNA triplex stabilising capacity being extraordinary; when the DNA triplex is formed in a 0.1 M NaCl buffer, its triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5oC upon drug association. The results indicate that 9-OH-B220 has the potential of being used both as a partner in an antigene strategy and as an antiretroviral agent.An NMR study of the base-pair breathing dynamics of hybrid duplexes formed between DNA and the nucleic acid analogue PNA revealed intriguing kinetic features of these complexes. PNA strand bases open and close with unusually high rates. The bases in the complementary DNA strands are influenced by this fast kinetics in different ways; while the DNA strand guanines are virtually unaffected, the thymine imino protons become hypersensitive to exchange catalysis. Hence, we conclude that base-pair opening is an asymmetric process in these hybrid duplexes. A model compatible with experimental data, in which a longitudinal breathing motion within the backbones is a pre-equilibrium state to that of lateral base opening, is presented and discussed. The results are of importance for efficient development of new DNA modulating drugs.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biofysik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biophysics (hsv//eng)

Nyckelord

Nucleic acids

binding modes

polarised light spectroscopy

linear dichroism

NMR

triple helix

base-pair breathing dynamics

DAPI

porphyrins

quinoxaline derivatives

Biophysics

Biofysik

Biophysics

biofysik

Publikations- och innehållstyp

vet (ämneskategori)

dok (ämneskategori)