(343b) Mammalian Cell-Compatible "Light-up" RNA Aptamer Biosensors for Custom Small Molecule Targets

In vivo small molecule imaging is an exciting area in biosensor research. Molecular imaging has applications in investigating and visualizing biological processes, screening and diagnosis, personalized medicine, and drug development. Currently a variety of methods are used both clinically and pre-clinically including CT, PET, SPECT, MRI, and ultrasound. Additionally, bioluminescence, fluorescence, photoacoustic, and Raman based techniques are also used in research efforts. Still, improvements to spatial, temporal, and molecular specificity and sensitivity are desired.

The use of aptamers poses a potential solution by using single-stranded DNA or RNA oligonucleotides capable of binding to specific molecules. Efforts to use fluorescent aptamers for direct small molecule sensing were pioneered with the development of Spinach, an RNA aptamer mimic of GFP (green fluorescent protein). Spinach was subsequently converted to a fluorescent small molecule sensor by inserting naturally-occurring riboswitch motifs into the parent fluorescent RNA structure.

Spinach was originally generated using standard Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approaches to isolate aptamers that could bind the DFHBI chromophore. A revisit to the SELEX process was used to select newer aptamers via expression of RNAs within E. Coli cells and fluorescence-activated cell sorting (FACS). This method was used to create Broccoli, a markedly improved aptamer with nearly twice the brightness of Spinach, enhanced thermostability, and ion biocompatibility.

Overall, despite these advances in the use of fluorescent aptamers for molecular detection in mammalian cells, a persistent limitation still exists with the small molecule recognition motifs. Specifically, recognition motifs to this point have all been derived from naturally-occurring riboswitches, which limits the applicability of these techniques to small molecules with cognate riboswitch pairs. This limitation also completely eliminates the use of aptamer biosensors for non-native small molecules like synthetic drugs. SELEX techniques have been developed to generate aptamers with riboswitch-like activity. For example, a technique called capture SELEX selects for conformational changing aptamers. However, such SELEX techniques for generating structure-switching motifs have not been combined with aptamer biosensor workflows, which would enable new capabilities in this space.

Here, we sought to develop an integrated SELEX approach to address this issue. We focused on l-3,4-dihydroxyphenylalanine (L-DOPA), a normally occurring amino acid precursor to the neurotransmitter dopamine, for which an existing riboswitch does not exist or has not been identified. We used capture SELEX to generate RNA candidates that could prospectively bind L-DOPA. We inserted a subset of candidates into the Broccoli construct and tested fluorescence in transfected HEK cells in the presence and absence of L-DOPA. These tests led to the identification of aptamer 3F, which exhibited modest light-up activity in cells. Live cell imaging was performed on Caco2 cells transfected with 3F in the same expression system. Caco2 cells differ from HEK cells in that they have the enzyme dopa decarboxylase responsible for the metabolism of L-DOPA. This enzyme can be inhibited by carbidopa. 3F transfected Caco2 cells given L-DOPA and DFHBI steadily lost fluorescence over time while those treated with carbidopa maintained fluorescence 45 minutes later. In general, L-DOPA metabolism causes fluorescence to decrease while inhibition of this degradation pathway maintains higher L-DOPA concentration and fluorescence. This data suggests that 3F fluorescence is dependent on L-DOPA concentration in mammalian cells. To overcome inefficient transfection efficiencies, we created a stable 3F-expression Caco-2 cell line and carried out detailed characterizations of L-DOPA biosensing dynamics. Overall, our results establish a strategy for creating mammalian cell-compatible RNA biosensors against custom small molecule targets.