(520g) Engineering DNA-Based and Protein-Based Materials for Live Single Cell Analysis

Cells are primarily comprised of metal ions, small molecules, proteins, lipids, and nucleic acids. The ability to probe these molecules in single living cells can shed new light on chemical processes inside of cells or allow disease diagnosis based on molecular profiling. However, there exists a lack of tools that allow one to monitor and analyze these molecules dynamically in live cells. Although genetically-encoded fluorescent tags have transformed live-cell protein analysis, there is a deficiency of robust techniques for studying other molecules. In this regard, probes based on nucleic acids have recently emerged as powerful tools for studying intracellular processes. Their biocompatibility, amenability to genetic encoding, low cost, ease of synthesis, modular structure, and ability to be chemically modified in a sequence-defined manner make them especially useful in sensing applications. By tuning their sequence, nucleic acids can be designed to recognize a wide range of molecules including other nucleic acids, proteins, ions, and small molecules.

The earliest analysis techniques based on DNA, such as in situ hybridization and polymerase chain reaction (PCR), required the fixation and lysis of cells, respectively, preventing their use for live-cell analysis. The introduction of linear DNA probes that can study events in live cells, such as molecular beacons, helped to expand the capabilities in the field. However, such linear DNA probes do not efficiently cross the cell membrane without the use of transfection reagents, and they are susceptible to rapid nuclease degradation in the cellular environment.

To overcome these challenges, NanoFlares were developed in 2007 as a new tool for live-cell analysis. NanoFlares are comprised of a gold core functionalized with recognition strands (hybridization-based, aptamer, DNAzyme, or aptazyme) for a target of interest. These recognition strands are hybridized to short fluorophore-labeled flare strands. Close proximity between the gold nanoparticle and the fluorophore quenches the fluorescence. When the target is present and binds to the recognition strand, the flare strand is displaced, separating the fluorophore and gold, and turning on fluorescence. Owing to the dense orientation of DNA on the nanoparticle surface in a spherical nucleic acid (SNA) architecture, NanoFlares have high cellular uptake without the need of transfection reagents, display enhanced resistance to nuclease degradation in comparison to free nucleic acid probes, have enhanced target recognition and binding, and exhibit little immunogenicity or toxicity. To date, NanoFlares have been used in over 50 studies for studying various targets including mRNA, small molecules, ions, and proteins.

Although NanoFlares constituted the first platform for live intracellular analysis at single-cell resolution, challenges still exist. These challenges include false-positive signal due to non-specific separation of the fluorophore and gold nanoparticle quencher, limited quantitative capabilities, inability to spatiotemporally track analytes, loss of binding affinity for the target due to partial blocking of the recognition strand, and the restriction that only targets with known nucleic acid-based recognition sequences can be detected. In this presentation, I will discuss the development of a new class of live cell probes via quencher free signal transduction that overcome these limitations and enable the study of a variety of intracellular analytes with high sensitivity and specificity.

In the first part of the talk, I will describe the discovery of a new class of signaling aptamers called “Forced intercalation (FIT)-aptamers.” FIT-aptamers consist of a viscosensitive dye as a nucleobase surrogate. Target binding to the FIT-aptamer induces a conformational change, forcing the dye into a more rigid (“viscous”) environment and turning on its fluorescence. I have shown that this is a general signal transduction strategy that can be coupled to the most common aptamer-target binding modes, capable of detecting ions and proteins at nM sensitivity in complex media. Moreover, I have shown that this fundamentally new strategy for interfacing aptamers with a readout event holds several key advantages over conventional approaches. Because this method is quencher free, it is resistant to false-positive signal. Furthermore, FIT-aptamers are kinetically superior to displacement-based strategies that require partial blocking of the aptamer site and show higher signal-to background ratios than traditional FRET-based techniques. I will end this portion of the talk by discussing our recent work with the Air Force Research Laboratory applying FIT-aptamers as a new tool for studying steroid hormones in clinical serum samples. Taken together, FIT-aptamers constitute the only light-up aptamer probe that can detect a wide variety of analytes with only a single modification.

In the second part of the talk, I will discuss the use of FIT-aptamers as the recognition element for the construction of next-generation NanoFlares. This new design holds several key advantages over first-generation gold NanoFlares: reduced false-positive signals, ability to utilize biocompatible and/or functional cores as a gold quencher is no longer required, and the ability to monitor intracellular analytes with spatiotemporal resolution. Using beta-galactosidase as the biocompatible core and pH-sensitive i-motif FIT-aptamers as the recognition moiety, I have successfully synthesized next-generation NanoFlares called FIT-Flares. I have shown that FIT-Flares do not elicit a false-positive signal in the presence of nuclease in buffer. In comparison, a gold i-motif NanoFlare has a 15-fold turn-on in fluorescence in the presence of nucleases due to non-specific separation of the fluorophore and gold nanoparticle. To verify that these results are translated within cells, I have also performed pulse-chase experiments. Studies with the MDA-MB 231 cell line show that scrambled (i.e. non-targeting sequence that should not turn-on in cellulo) gold NanoFlares show a steady increase in fluorescence signal as the chase time is increased, as measured by flow cytometry. In contrast, no changes are observed in the fluorescence of scrambled FIT-Flares as chase time is increased, further corroborating that non-specific signal is diminished in this new design. Importantly, pH-sensitive FIT-Flares are able to report changes in intracellular pH. Taken together, these experiments demonstrate that compared to gold-NanoFlares, FIT-Flares constitute a superior platform for monitoring intracellular analytes in live cells.

In the third part of the talk, I will describe how a FIT-strategy allows for increasing the scope of analyte recognition in cells. In particular, the use of a FIT-strategy allows one not only to detect analytes through the DNA shell but unlocks new capabilities by allowing the use of a functional protein core for detecting targets, thereby vastly expanding the range of analytes that can be detected. I hypothesized that by using a functional core such as an enzyme, I can detect analytes for which there are no aptamers with biologically relevant binding affinity. To test this hypothesis, I designed an SNA using glucose oxidase (GOx) as the core. To detect glucose intracellularly, I developed a new two-step assay. In the first step, cells are treated with GOx SNAs in glucose-free media. GOx catalyzes the conversion of glucose to gluconic acid, with production of hydrogen peroxide. In the second step, the cells are treated with fluorescein bis benzyl boronic ester (FBBBE), a cell permeable, non-fluorescent boronate-ester of fluorescein. In the presence of hydrogen peroxide, the boronate groups are cleaved and highly fluorescent fluorescein is formed which is retained intracellularly. Therefore, the fluorescence is directly proportional to the amount of glucose in the cell. This new strategy affords greater than 100-fold fluorescence turn-on in buffer, discriminates between glucose and its close analogs (i.e. glucose-6-phosphate), and can detect glucose in 9 different cell lines. Taken together, protein-based FIT-Flares constitute a plug-and-play platform that can make measurement inside of living cells using either binding-based sensing (using the DNA shell) or activity-based sensing (using the protein core).