Results and Discussion: Our pursuit of live cell RNA analysis probes was motivated by the advances of T cell therapies. We investigated whether it could be possible to distinguish sets of T cells based on expression of different T cell receptors and envisioned fluorogenic hybridization probes targeting the hypervariable CDR3 region. This region determines the TCR specificity and varies across different T-cell lines. We commenced our study by preparing DNA FIT probes (Fig. ) targeting the CDR3 regions of TCR mRNA in the CCRF-CEM T cell line. The serinol-linked QB nucleotide SQB (see Fig. ) was introduced at a central position of oligonucleotide probes. As previously described, SQB was flanked by an LNA nucleotide. During DNA synthesis, alkyne-modified uridine was coupled at varied distances to the serinol nucleotide. The alkynyl groups were used as conjugation handles for post-synthetic introduction of auxiliary dyes. We selected the sulfonated AF 568 dye and assumed that the net negative charge would minimize interactions with negatively charged DNA/RNA. In the last step, the QB dye was introduced by coupling to the amino group of serinol as previously described. Hybridization of the QB-containing FIT probe C1-QB with the RNA target was accompanied by a 60-fold increase in fluorescence (Fig. ). For comparison, we prepared probe C1-AF568, which tethers an AF568 dye to the 5-position of a uridine. This probe is much brighter than C1-QB, but non-responsive (Fig. , Table ), indicating efficient dye solvation in both single and double stranded states. Of note, introduction of the QB dye in C1-AF568-QB rendered emission from AF568 responsive to hybridization, as shown by the low fluorescence of the single stranded probe (Fig. ). The absorption band at 550 nm indicates dye-dye contact in the absence of target RNA. In the target-bound state, dual labelled probe C1-AF568-QB is brighter than the mono-labelled probe C1-QB (Fig. , right). The quantum yield (Table ) remained at the level of the C1-QB probe indicating that excitation energy is efficiently transferred from AF568 to QB with little, if any, contact quenching. We conclude that bright emission of target-bound C1-AF568-QB results from a combination of extinction coefficients of the dye constituents. Similar properties were observed for probes in which AF568 was separated from the QB hybridization reporter by 2, 4 or 8 spacer nucleotides (C2-, C3-, C4-AF568-QB, Fig. , Tables and). The added extinction coefficients of the two dyes ensured an enhanced brightness of the probes while maintaining low single-strand fluorescence due to QB-induced quenching of AF568 emission in absence of target. A comparison of the four probes shows that fluorescence turn-on is increased with decreasing AF568-QB distance (Fig. , left). The accompanying decrease in single strand fluorescence (Table ) suggests that proximity facilitates quenching of AF568 by QB. Conversely, brightness increased with increasing distance between the two dyes, which we attribute to a reduction in energy transfer efficiency.