Hybridization probes that fluoresce upon binding to specific nucleic acids offer instantaneous detection of RNA and DNA targets without the need for downstream analysis of hybridization mixtures. Furthermore, monitoring a specific RNA in living cell offers possibility to gain understanding of its functions, trafficking, and localization in subcellular environment1,2. Spinach aptamer is an RNA that binds 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its fluorescence by ~80 times. The property that can be used to monitor RNA as green fluorescent protein (GFP) is used to monitor proteins in live cells1,2. In this study we used the recently published structure of Spinach aptamer3, and designed a split Spinach aptameric (SSA) probe for detection of nucleic acids. The parent aptamer structure was divided into two parts. Nucleic acid binding arms were added to each part through triethylene glycol or dinucleotide linkers. Binding of the split probe to a specific analyte re-formed the Spinach DFHBI-binding site3 thus enabling DFHBI fluorescence. After 60 min of incubation, fluorescence increased c.a. 64-76 times, with limit of detection ~1-2 nM. Since one analyte binding arm of SSA is relatively short, single mismatched base pair in analyte greatly destabilizes the hybridization4,5, leading to loss of fluorescent signal. SSA dedifferentiated against a single base mismatched DNA analyte at room temperature in ~30 sec after mixing and maintained high differentiation for over 4 hours. SSA probes designed and tested in this study can be used both for visualization of specific RNA in live cells and for in vitro analysis of nucleic acids e.g. for testing mutations and single nucleotide polymorphisms responsible for genetic diseases, cancer and drug resistance in pathogenic bacteria and viruses.

Aptameric sensor for fluorescent analysis of specific nucleic acid sequence

253rd National Meeting (2017)

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