The effects of DNA interacting drugs within cells can be experimentally observed at many different levels (Figure 1). The first, most basic level, is the chemical interaction of the drug with the DNA double helix. A variety of techniques have been developed to examine this level of interaction, and have shown that the chemistry of the interaction is very similar if studied on naked DNA or in cells in culture. Alkylation at specific sites and sequences in cells can be examined by using a modified thermal cleavage assay. Following reaction of cells with the DNA alkylator, genomic DNA is extracted and heated to induce breaks at alkylation sites. The frequency of breaks can be ascertained by measuring the presence of modified nucleotide residues using HPLC or mass spectrometry. Time course experimentation can be performed to determine how the DNA adduct is maintained in the context of the nuclear environment. The site of alkylation and preferred sequence can be examined in cells using PCR stop assays wherein the DNA adduct prevents Taq polymerase elongation, or by ligation of the broken DNA with linker DNA molecules, followed by site-specific PCR and analysis of recovered products [3-7]. Compounds that bind tightly but reversibly to DNA can be analysed for their binding sites by footprinting assays, where short, defined DNA sequences (100–400 bp) are treated with random DNA-breaking agents in the presence of the compound, which protects from cleavage at its preferred binding sequences. This technique has been extensively used to study minor groove binding drugs such as distamycin A and the bis(benzimidazole) Hoechst 33258 [8-12].
A second degree of interaction is the disruption of DNA/protein or other nucleic acid interactions (such as important DNA secondary structures) that result from the drug/DNA interaction. Transcription factors, chromatin-associated proteins, and proteins involved in replication, recombination and repair are examples of proteins that recognize aspects of the DNA double helix (major versus minor groove, B versus non-B form DNA), certain DNA sequences and regions, or other DNA modifications, all of which could be affected by drug binding. The drug/DNA interaction might stabilize a transient DNA structure, introduce a new conformation or structure, or mask a DNA modification and in these ways alter the normal regulatory processes working at these sites [13-15]. Some of the proteins that have been examined include the high mobility group (HMG) proteins [16-20]. These chromatin-associated proteins induce DNA bending, recognize distorted DNA structures, and play a role in regulating gene expression, repair and recombination. Electromobility shift assays (EMSA) [21-25] and chromatin immunoprecipitation (ChIP) [26-30] are some of the methods used to assess DNA/protein interactions in vitro and in vivo. Expression microarrays and real-time reverse transcriptase PCR can be used to assess changes in transcription of genes in the presence of DNA-binding compounds, a likely end product of alterations in protein/DNA binding. Indeed, transcription of 21 genes with promoters containing match sites for the minor groove-binding hairpin polyamide/chlorambucil conjugates were significantly inhibited in the presence of these compounds.
DNA secondary structures, such as those that form during DNA recombination, or G-quadruplexes found at the ends of chromosomes and sometimes present within regulatory regions of genes, are other examples of possible targets for drug design. Several recent reviews have discussed some of these structures and their relevance for targeting[13,31,32]. Techniques that are used to investigate these DNA structures, their orientation and polarity, and stability in vitro include CD, IR, and NMR spectroscopy, X-ray crystallography, and molecular techniques such as native gel electrophoresis, chemical probing of isolated DNA species with nucleotide-specific cleavage reagents, PCR and primer extension reaction stop assays [33-37].
More global changes in the nuclear environment can be visualized using a combination of fluorescence in situ hybridization (FISH) and confocal microscopy techniques,"ImmunoFISH", a relatively new technology that allows coincident visualization of protein complexes and DNA in intact nuclei[38,39]. This type of microscopy enables visualization of nuclear complexes composed of DNA and protein, such as nuclear factories, the nuclear matrix attachment sites and organizational centres for processes of DNA replication and transcription. Alterations in these complexes that result from drug treatment might not be detected using other molecular techniques. This technology is particularly useful for examining the nuclear distribution of specific genomic locations or sequences which might change during the course of the cell cycle. For instance, this technique has recently been used to examine distribution and binding of membrane Tel binding protein (MTBP/TRF2) to telomeres during spermatogenesis and the cell cycle. Deconvolution microscopy using fluorescently-labelled polyamide compounds has also been used to monitor drug uptake and cellular distribution . These technologies will undoubtedly provide insight into the global effects of drug treatments and aid in future drug design.