Hybridization probes that fluoresce upon binding to specific nucleic acid sequences offers instantaneous detection of specific RNA and DNA targets without the need for downstream analysis of hybridization mixture. Spinach is RNA in complex with fluorophore that mimics enhanced green fluorescent protein (GFP) and emits green fluorescence comparable to that of fluorescence proteins1,2. Chemically synthesized fluorophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is nonfluorescent but upon binding to Spinach, it gives enhanced fluorescent quantum yield of 0.72. Based on the folding structure of recent crystallized Spinach aptamer3, Spinach was separated in two strands by removing the stem loop. The 5’ end and 3’end was shortened in order to reduce the association of the two strands when free in solution. Nucleic acid binding arms were added to 5’ end and to 3’ end through peg linker. We term this complex as “split-Spinach Aptamer (SSA)”. The main role of the binding arms is to re-form the Spinach internal bulge of G-quadruplex where the fluorophore binds3 by hybridization to the complementary DNA analyte and allowing the DFHBI to be fluorescent. Since one nucleic acid binding arm is relatively short compared to the arm on the 5’ end, single mismatched base pair on the DNA analyte will greatly destabilizes the hybridization4,5, leading to destabilization of the SSA. We demonstrated the high selectivity of SSA towards complementary DNA analyte and mismatched DNA analyte through real time detection at room temperature. After 60 min of incubation, fluorescence increased c.a. 76 times with limit of detection 1.9nM. SSA differentiated single base mismatch in DNA analyte immediately after incubation. Funding from NSF CCF (1117205 and 1423219), and NIH R15AI10388001A1 is greatly appreciated

Binary aptamer for instant fluorescent analysis of specific nucleic acids in vitro

251st National Meeting (2016)

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