Enzyme-free and multiplexed micro RNA detection using micro RNA-initiated DNA molecular motor

In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.