Our research was primarily concerned with oligonucleotides and their application as pH probes in cells. Accurate methods of in-vitro pH monitoring are needed to improve the efficacy of oligo-based drug delivery. To explore this, my mentor was working with fluorescein, one of the oldest and most widely used fluorophores in biological research. Fluorescein’s quantum yield also depends on pH because of the equilibrium between its two resonance structures within pH 4.0-7.5. Consequently, the user can measure fluorescence with changes in the pH to monitor the molecule’s environment.

The pH sensitivity of FAM (6-carboxyfluorescein) attached to an oligonucleotide has been demonstrated, however it lacks the sensitivity for use in live cells. According to a paper exploring fluorescence in peptides, the thiourea linker in FITC (fluorescein isothiocyanate) quenches fluorescence. Applying this to the previous FAM experiment and observing a parallel effect with an oligo pH probe could enable live cell use.

A 5 nucleobase FITC labeled oligonucleotide (5’-FITC-[2’-OMe]U 5 -3) was synthesized and purified. Synthesizing the FITC labeled oligonucleotide caused byproduct reactions that obstructed purification, so confirmation that the intended product was created was achieved through secondary ion mass spectroscopy conducted by Novatia LLC. The FITC oligo was then suspended in McIlvaine buffer ranging from pH 4.05 to pH 7.73. The fluorescence was then measured with an M1000 imager and compared to the previous FAM experiment. Plotting fluorescence as relative fold change, where each pH value is compared to the lowest demonstrates that the FITC labeled oligo exhibited approximately double the fluorescence quenching when compared to the FAM labeled oligo (~41.7 vs. ~21.3).

As hypothesized, the linker caused the increase in quenching, however future experiments could confirm this. In addition, it would be beneficial to improve the synthesis and yield, which is likely why FITC labeled oligonucleotides are not commercially available. Finally, using this result and applying it to monitor pH and therefore cargo uptake in live cells is a potential future direction for inquiry.