https://doi.org/10.15255/KUI.2003.018
Published: Kem. Ind. 52 (11) (2003) 545–552
Paper reference number: KUI-18/2003
Paper type: Conference paper
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Methods for Study of Noncovalent Interactions of Small Organic Molecules with DNA and RNA. More Detailed Overview of Intercalative Mode of Binding.

I. Piantanida

Abstract

A number of compounds are designed and prepared with the aim of biological and/or biochemical implementation (therapeutic usage, markers, molecular devices...). Study of noncovalent inteactions of small organic molecules with natural and synthetic polynucleotides in aqueous media is a logical choice for fast screening after preparation of novel DNA and RNA active substances. Today, a number of rather simple and handy experimental methods is available for the study and characterisation of the complexes between the polinucleotide and a small molecule of interest. The bases of RNA and DNA are active chromophores. The thermal denaturation of nucleic acid can be monitored by measuring the change in UV absorbance of a nucleic acid solution as a function of temperature. A significant hyperchromicity (a higher molar extinction coefficient) of the nucleic acid at 260 nm is observed upon the melting of duplex regions into single stands. The temperature of melting (Tm) is defined as the temperature where 1/2 of the DNA or RNA becomes denatured; for duplex nucleic acids this transition is highly cooperative and appears as an inflection point in the plot of absorbance versus temperature. The Tm can be monitored in the presence of small molecule ligands to examine relative binding energies. In most cases, the small molecule binds to the duplex nucleic acid with a higher affinity than to the sin- gle-stranded form, thus stabilizing the duplex form. Stabilization shifts the Tm to higher temperatures. The change in the Tm (ΔTm) is proportional to the ligand's affinity to the folded form of nucleic acid weighed by its affinity to the melted form. UV-VIS absorbance and/or fluorescence emision spectroscopy are routinely used to probe RNA or DNA binding of small molecules. The sensitivity of each technique is, however, significantly different. Fluorescence emision measurements are often 100 -1.000 fold more sensitive than absorbance spectroscopy. By measuring the spectral changes of the ligand as a function of nucleic acid concentration, binding isotherms can be generated and used to calculate affinities. Analysis of direct binding data must take into account the concentration of the observable species relative to the affinity of the small molecule -DNA/RNA interaction being studied. For most titrations, the concentration of the observable species is kept constant, while the non-observable species is titrated in. If the concentration of the observable species is much lower than the Kd of the interaction, then the binding isotherm is sensitive only to the highest affinity binding site. If the binding interaction is non-cooperative, direct binding data can be analyzed using a Scatchard plot. This plot can simultaneously determine, both, binding stoichiometry and the average affinity of each site. A Scatchard plot is made by plotting r versus r/Cf. The slope of this plot will equal Keq, and the X-intercept will equal the number of binding sites. Large errors with Scatchard analysis are often encountered. The data points for the 100 % free and the 100 % bound states are "weighed" much more heavily than the points in the middle of the titration. Non-linear analysis of binding data can help reduce the errors asociated with quantifying the spectral properties of these "extreme" (and often inaccurate) data points. Line shape analysis can eliminate much of the error asociated with quantifying the spectral properties of the 100 % "free" versus the 100 % "bound" states of the observable species. Non-linear analysis typically weighs all data points equally and fits all the points to a theoretical curve. The fluorescence intensity of ethidium bromide typically increases upon the binding of nucleic acids. Subsequent displacement of ethidium is apparent by a decrease in emision intensity upon addition of a competitive inhibitor. Since it binds to most DNAs and RNAs, ethidium displacement experiments can be conducted using a wide range of nucleic acids. The concentration of competitive inhibitor needed to displace 1/2 of the ethidium bromide is marked as IC50. However, direct binding isotherms are more thermodynamically meaningful than activity measures (IC50 values). Methods mentioned in this text are simple to learn and unexpensive to handle. Results obtained from these methods allow the determination of eventual selectivity or specificity of studied compounds and comparison with the previously studied analogues. This methodological approach is especially suitable for the systematic study of the large series of structuraly close analogues. Results obtainied for the large series of analogues often allow the choice of the most attractive substances for the biological studies, in this way saving time and material.

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Keywords

DNA, noncovalent interactions, intercalation, binding constants