Background free amplified single-molecule FISH for in situ and flow cytometric applications

Abstract Cellular RNAs can serve as powerful biomarkers of diverse disease states. Recent developments in probe technology enable the detection of RNAs with single-molecule sensitivity by fluorescence in situ hybridization (sm-FISH). This technology holds great promise in clinical diagnostics for the detection of pathogens and cancers. However, its utility for imaging pathological sections is presently limited, because in order to obtain single-molecule sensitivity, imaging needs to be performed with high magnification objectives that have a limited field of view, permitting the observation of only a few cells at a time. The relatively low intensity of these signals also limits the utility of sm-FISH when the cells are analyzed by flow cytometry, because rare cells, such as immunological memory cells or stem cells, which express a low level of mRNA markers, cannot be detected. A further limitation is that single nucleotide variations cannot be detected. We are developing a new generation of sm-FISH in which the signals is amplified without any amplification of background. In our approach, that we refer to as strictly target-dependent amplified FISH (stamp-FISH), when a pair of binary probes bind to the target a sequestered sequence is exposed. The exposed sequence then serves as an initiator of a hybridization chain reaction, which leads to creation of a large, highly fluorescent DNA cluster that remains tethered at the target. We obtain as much as 10-fold amplification of signal without any enhancement in background. Furthermore, the probes yield exquisite discrimination between single nucleotide variations. We will develop this technology further and apply it for detection of point mutations in EGFR and BRAF mRNAs. Currently, about fifteen percent of the copies of target mRNA that are present within the cells can be detected. We will improve this efficiency using strategies aimed at increasing the binding of probes to the target mRNAs. Probe sets will be developed for an exemplary set of mutations within the EGFR and BRAF genes and then cell lines and cancer tissues, in which these mutations are expressed, will be imaged using these probe sets. We will also develop multiplex assays that will detect three mutants and the wild-type mRNA simultaneously. In addition, for T cell profiling we are proposing to detect 15 RNA markers in multiplex in T cells. With this expansion of the multiplexing range to 15 targets, we will be able to detect subtypes of cells that differ from each other by the expression of chemokine/cytokines, transcription factors, or metabolic markers. This development will create powerful new analytical possibilities for diverse fields in both research and clinical settings and thus will be of broad utility.