ABSTRACT
Various fields in biomedical research, drug discovery, and disease diagnostics call for methods allowing the sequence-specific detection of DNA or RNA molecules. Recent developments in sequencing technologies have led to dramatic reductions in cost and sequencing time (Guo et al. 2008; Mardis 2008; Clarke et al. 2009). The widely used sequencing infrastructure has prompted the discovery of an ever-growing number of DNA and RNA targets with hitherto unknown function. These discoveries increase the need for chemical probes that allow the analysis of the expression of newly discovered target genes in real time and within living cells. Conventional methods of DNA/RNA detection such as quantitative polymerase chain reaction (qPCR) (Saiki et al. 1985), northern blotting (Alwine et al. 1979), and DNA-microarray technologies, as well as sequencing, provide a general overview of the ensemble of the collected cells (Schena et al. 1995; Ooi et al. 2001; Swain et al. 2002; Raser and O’Shea 2004), which need to be harvested before analysis. Sample collection, preparation, and analysis often take several hours and are complicated by low sample stability (e.g., affected by nucleases), contamination, and the loss of a considerable amount of sample material. Fluorogenic oligonucleotide probes enable direct measurements within living cells. Imaging experiments allow the visualization of gene expression dynamics and reveal details about the transport and degradation of RNA molecules. Perhaps most importantly, RNA imaging experiments address the complex interplay between the target and the multitude of biomolecules present within the biological matrix and thereby provide insight into the cellular functions of RNA.
