Session: Current Topics in Biological Chemistry:

The ability to probe the composition of live cells is important for learning about chemical processes inside of cells as well as for creating diagnostic platforms based on molecular profiling. While fluorescent proteins and nucleic acid-based aptamers have revolutionized live cell imaging, these sensors must be genetically encoded into the cells and therefore have limited applications in a clinical setting. Many techniques require cell lysis or a bulk population of cells to yield a detectable quantity of the target analyte. Additionally, a major impediment to the development of intracellular probes is the inability of most probes to traverse the cell membrane. Consequently, these probes require the use of transfection reagents or physical methods that perturb the cell membrane, which in turn can interfere with the measurements by changing the analyte’s expression profiles. Furthermore, existing strategies for signal transduction to indicate the presence of a given analyte, such as fluorophore-quencher-based systems, are limited and susceptible to false-positive signals.

To address these challenges, I have first developed DNA-based aptamers that utilize a novel sensing strategy. Specifically, these aptamers are chemically modified at a single base with a viscosity-sensitive dye. Target binding to the aptamer changes the conformation of the aptamer and forces the dye to intercalate (or FIT) between the DNA base pairs, turning its fluorescence on. This “FIT” strategy avoids false-positive signals common to most fluorophore-quencher-based systems, quantitatively outperforms FRET-based techniques, gives up to 20-fold fluorescence enhancement in buffer, and allows for the detection of analytes in complex biological milieu such as human serum.

To apply this technique to the analysis of intracellular analytes, I next developed a new platform based on protein spherical nucleic acids (ProSNAs). ProSNAs are composed of a protein core surrounded by a DNA shell. The dense functionalization and radial arrangement of the DNA around the protein core allows efficient cellular uptake of the ProSNA without the use of transfection reagents. Additionally, using a FIT-aptamer as the DNA sequence allows for the detection of intracellular analytes. As an example, I demonstrate that using an i-motif sequence that undergoes pH-dependent conformational changes allows for the detection of intracellular pH levels.

Caroline Kusmierz

Appearances