Fluorescence-Based Methods for the Elucidation of RNA: Small Molecule Interactions
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Although noncoding RNAs are recognized as important cellular regulators that may be misregulated in diseases, methods to investigate RNA secondary structure recognition have been limited. To help establish guiding principles for understanding RNA conformations, we introduce fluorescence-based methods to demonstrate that different RNA secondary structures can be differentially recognized by libraries of small molecules. An environmentally sensitive and optically enhanced benzofuranyluridine (BFU)-phosphoramidite fluorophore was first synthesized and incorporated into a library of RNA secondary structural motifs to observe aminoglycoside-RNA interactions. Using Principal Component Analysis (PCA), we demonstrated that five canonical RNA secondary structural motifs can be significantly discriminated with 100% predictive ability. Efforts to further distinguish motifs based on size/sequence by varying assay salt concentrations demonstrated the importance of RNA conformational dynamics and flexibility for improving the assay’s predictive power. Furthermore, an RNA recognition-based approach was implemented to demonstrate that the 3’ Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT-1) triple helix – a biologically relevant and unique structure – can be differentially recognized by a set of diphenylfuran (DPF) scaffold-based small molecules. Taken together, these developed methods illustrate the connection between RNA secondary structure and its molecular recognition.
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