Biophysical studies on the interaction of polyamines with nucleic acids

The biogenic polyamines spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine(CAD) are ubiquitous linear organic molecules present in all eukaryotic and prokaryotic cells. Theyare thought to be essential for the normal functioning of the cells, like cell growth and play essentialroles in a wide variety of cellular regulatory pathways and functions like gene regulation, DNA packaging, proliferation etc. The binding of biogenic polyamines SPM, SPD, PUT, CAD and theanalogue 1-naphthyl acetyl spermine (NASPM) to different natural DNAs, synthetic DNAs anddouble stranded RNAs has been investigated in this thesis.The comparative binding studies of biogenic polyamines SPM, SPD, PUT and CAD with CT DNA proved the binding to be strongest for SPM. Conformational studies revealed perturbation of DNA structure on binding of the polyamines; the gross B-form of DNA, however, remained more or less unaltered. Thermal stability of DNA was remarkably enhanced on binding and the binding constants derived revealed highest affinity for SPM. Interaction of the polyamines were endothermic and entropy driven. The binding affinity of SPM (6.20105 M-1) was much higher than those for the other polyamines. Small but negative heat capacity changes in the four cases suggested the involvement of significant hydrophobic forces in the complexation. Overall, the affinity varied as SPM > SPD > PUT > CAD. The comparative binding study of these polyamines with three natural DNA of different base composition i.e. Clostridium perfringens (CP) DNA (27% GC), Escherichia coli (EC) DNA (50% GC) and Micrococcus lysodeikticus (ML) DNA (72% GC), revealed significant changes in the conformation of each of the DNA, but was higher with the DNA with high AT content. Maximum thermal stabilization was shown by CP DNA followed by EC and ML DNAs. The lower IC50 values (concentration of polyamines required to quench fluorescence of ethidium bromide-DNA complex by 50%) in terms of ethidium displacement and higher binding affinities varied as SPM> SPD> PUT> CAD. The positive entropy term was higher in the CP DNA compared to the other two DNAs confirming more displacement and release of the water of spine in the minor groove of the AT rich DNA. The values of standard heat capacity change obtained for the systems also supported a groove binding model. Enthalpy-entropy compensation was observed in each system studied. The polyelectrolytic contribution to the Gibbs energy decreased with the increase in salt concentration, whereas, the non-polyelectrolytic contribution was not affected which showed that electrostatic interaction played a remarkable role in the binding of polyamines to DNA. Thus, polyamines bound stronger with AT rich CP DNA and least with the GC rich ML DNA and the binding to each DNA was stronger for SPM and varied as SPM> SPD> PUT> CAD. The results proved the AT base specificity of the polyamines.The binding of the polyamines, SPM, SPD and the polyamine analogue NASPM with synthetic DNA polynucleotides of different base sequences viz. poly(dA).poly(dT), poly(dA-dT).poly(dAdT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) revealed significant conformational perturbations in all the four polynucleotides. Binding of SPM, NASPM, SPD to all the 2 polynucleotides was driven by large positive standard molar entropy changes, clearly revealing entropy driven binding. The strong positive entropy term in these interactions is suggestive of the disruption and release of water molecules from the grooves of the DNA thereby facilitating groove binding. Higher Go hyd values for the AT polynucleotides indicated that the hydrophobic contribution in the polyamine-AT polynucleotide complexes are much larger as compared to the GC polynucleotides. Thus, even though salt dependent studies revealed electrostatic interaction to be a prominent contributing force to the Gibbs energy, hydrophobic interaction also apears to play a role in the interaction. The binding affinity of SPM and SPD varied as poly(dA).poly(dT) > poly(dAdT). poly(dA-dT) > poly(dG).poly(dC) > poly(dG-dC).poly(dG-dC). The binding affinity of NASPM varied as poly(dA-dT).poly(dA-dT) > poly(dA).poly(dT) > poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC). Trend in the binding affinity was further confirmed from the binding affinities calculated from the melting stabilization (Tm) values obtained from optical melting and differential scanning calorimetry studies and was also corroborated from ethidium bromide displacement assay. The results revealed sequence selectivity of polyamines towards the AT sequences over the GC sequences. Furthermore, it proved the ability of NASPM to bind with the AT hetero polynucleotide with a higher affinity than the other biogenic polyamines. Thus, the results suggest importance of polyamine analogues and its ability to interfere with normal polyamine interactions as a method of cytotoxic activity. RNA targeting is an evolving new approach to anticancer therapeutics and in this context, the capability of polyamines and analogues to target the double stranded RNAs poly(I).poly(C), poly(C).poly(G) and poly(A).poly(U) has been studied to understand the structural and thermodynamic basis of the binding and the comparative efficacy of the analogue over the the natural polyamines. Circular dichroism spectroscopy revealed structural perturbations for the RNA polynucleotides on binding of the polyamines. Thermal melting results showed enhanced stabilization which varied as SPM > NASPM > SPD. Microcalorimetry results revealed the binding affinity to be strongest for poly(I).poly(C) and varied as poly(I).poly(C) > poly(C).poly(G) > poly(A).poly(U). The highest affinity was shown by SPM > NASPM > SPD. Ethidium bromide displacement assay reflected the strong ability of SPM to bind to the RNA sequences and corroborated the same trend. The strong positive entropy term in these interactions is suggestive of the disruption and release of water molecules from the grooves of the RNA thereby facilitating groove binding as suggested for DNAs. The interactions were characterized by total enthalpy–entropy compensation and high standard molar heat capacity values. The standard molar heat capacity change served as an indicator of dominant hydrophobic effect in the binding process. The binding was found to be influenced by salt concentration suggesting Go pe to be a significant contributor towards the Gibbs energy value.Atomic force microscopy experiments on the interaction of polyamines with double stranded RNA revealed significant morphological changes leading to compaction and condensation of the linear RNA molecules. Overall these studies provide an understanding of the underlying mechanism of polyamine interaction with nucleic acids that may enable us to approach a strategy for targeting the polyamine pathway as a means of antiproliferative mechanism