Chemical Imaging by NanoSIMS Provides High-Resolution Localization of the G-Quadruplex Interactive Drug (Br)-PhenDC3 on Human Chromosomes

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Determining thedistribution of biologically active compounds within cells is a major issue to understand their mechanism of action and to optimize their properties. Over the past decade DNA secondary structures called G-quadruplexes (G4) have been identified as key modulators of genomic functions. This very active research field has led to the development of G4-targeted molecular probes that are used to track quadruplex forming domains in cells, which is achieved, in most cases, by conventional fluorescence microcopy. However, the intrinsic low resolution of fluorescence microcopy as well as the necessity to tag the drugs with fluorophores represent strong limitations. Here we present the use of secondary ion mass spectroscopy imaging (nanoSIMS) for mapping within metaphase human chromosomes the distribution of a bromo-bisquinolinium phenanthroline derivative (Br- PhenDC3) used as G-quadruplex probe. In addition a statistical approach to increase the accuracy and the spatial resolution of the nanoSIMS imaging was implemented as a plug in for the image analysis software ImageJ. The results demonstrate the presence of Br-PhenDC3both at terminal and interstitial regions of chromosomes and constitute a demonstration of the effectiveness of nanoSIMS imaging as an alternative method for accurate genome-wide mapping of DNA interactive drugs.

Most anticancer chemotherapeutic agents used in the clinic as frontline drugs act as nuclear DNA binders. These agents are considered to bind more or less randomly on the polymeric structure of DNA, or at least in a non-controllable manner thereby hitting both crucial target regions and off-target regions. This uncontrollable distribution is assumed to be responsible for the high cytotoxicity and the potential mutagenicity frequently associated with DNA interactive drugs, two features often used as decisive arguments to decrease research and development studies on this class of compounds. Therefore, determining whether DNA drugs localize uniformly or show preference for certain genomic regions has become a crucial issue for the development of optimized DNA binding anticancer agents in the future. Surprisingly this topic remains largely unexplored so far, essentially due to the lack of genome-wide analytical methods. However this has been recently challenged by the emergence of Chemical-Sequencing (Chem-Seq) methodologies which propose to map the genomic distribution of drugs by identifying drug-induced DNA damages or repair protein recruitment using chemical capture and sequencing. Nevertheless, although powerful and highly promising, Chem-Seq approaches are still technically challenging, highly expensive in the case of whole-genome studies and require extreme caution in data analysis with stringent bioinformatics procedures [5,6]. In addition, they provide indirect read out and not direct visualization of drug DNA binding targets. Consequently, there is a strong need for new complementary imaging methods for identifying the distribution of DNA interactive drugs at the genomic level.

Although drugs can be labelled with fluorescent tags (or be intrinsically fluorescent) routine fluorescence microscopy provides resolution limited by light diffraction thereby enabling only the detection of spots (foci) corresponding to the presence of at least 20-40 fluorophores or more. This works fine for immunostaining strategies, in which the fluorescent signal is amplified by heavily labelled antibodies, but it is not applicable to the detection of small molecules unless these are confined in sub compartments (e.g. nucleus, mitochondria, lysosomes),which increases the density of fluorescent markers. Moreover, the labelling of drugs represents an issue as most fluorophores impact target recognition and may modify drug intracellular localization and penetration. Although super resolution microscopies hold great potential for chromosome and cellular imaging with high spatial resolution, they are far from being routine imaging techniques and they are highly dependent on the specific photophysics of the dyes.