RNAs play critical roles in most cellular and developmental processes. The RNA in vitro synthesis quantification method with low background without requiring translation is highly desirable. In this study, we have described a ratiometric RNA aptamer–fluorophore complex, Spinach2/DFHBI–CM, for quantifying the in vitro synthesis of RNA. DFHBI–CM exhibits bright fluorescence upon excitation at 337 nm with or without RNA, and displays the characteristic emission band of triazolyl-coumarin centered at 420 nm. When DFHBI–CM is combined with RNA aptamer Spinach2 to generate Spinach2/DFHBI–CM fluorescence complexes, under the excitation wavelength of 447 nm, the fluorescence intensity at 502 nm is significantly enhanced. By comparing the emission intensity at 420 nm and 502 nm, ratiometric fluorescence measurement is achieved. Based on this principle, DFHBI–CM was applied as a fluorophore in RNA aptamer–fluorophore complex for ratiometrically detecting and quantifying RNA in vitro synthesis. Our design has the following advantages: (1) ratiometric fluorescence can minimize the environmental inferring factors and thus, more accurate and effective detection can be achieved; (2) compared with run-off transcription detection of RNA, this ratiometric fluorescent probe reports immediately the quantity of RNA synthesized in vitro.
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