Studies Of Ultrasensitive Fluorescent Biosensor Based On Rolling Circle Amplification And Exonuclease Ⅲ-Aided Recycling

Rolling circle amplification (RCA) is an isothermal DNA amplification process with excellent capability of nucleic acid amplification. It can convert a short DNA primer into a long single-stranded DNA containing a large amount of tandem repeats that were the hybridization sequence of circle DNA template. The RCA products can be assigned different functions like drug loading, target recognition and fluorescence imaging by designed the sequence of circle DNA template. Therefore, RCA have been the subject of intense research in the past decades with its properties in chemistry, biology and clinical medicine. Exonuclease III (Exo III) also has been widely applied to the signal amplification strategies because of its specific recognition to the double-stranded DNA with abreast or recessed 3’end. In this paper, we take advantages of RCA and Exo III to construct a multiple amplification fluorescent biosensors for the detection of nuclease, micro RNA and small molecules.(1) In chapter 2, a robust fluorescent sensing platform for ultrasensitive assay of nuclease has been established based on rolling circle amplification and exonuclease Ⅲ-aided recycling amplification. The substrate DNA (sDNA) of S1 nuclease was designed as the primer to generate RCA products that can hybridize with the pre-quenched TaqMan probes and form recessed 3’terminus double-stranded DNAs. In the presence of exonuclease Ⅲ (Exo Ⅲ), the TaqMan probes were digested from the 3’-hydroxyltermini, releasing the fluorophore and generating enhanced fluorescence signals. Meanwhile, the RCA products with 3’protruding ends were liberated and hybridized with other TaqMan probes, triggering another cycles and obtaining remarkablely increasing fluorescence. However, in the presence of S1 nuclease, sDNA was cleaved into mono-or short-oligonucleotides pieces, which could not cause the RCA reaction and subsequent Exo Ⅲ-aided recycling amplification reaction, resulting in extremely weak fluorescence. The fluorescence intensity gradually reduced with increasing concentration of S1 nuclease. Due to the double signal amplification, the developed method was demonstrated to exhibit exceedingly excellent sensitivity with a detection limit of 5×10-7U μL-1. Moreover, the sensing system was used for the real sample analysis with satisfied results.(2) In chapter 3, a fluorescent sensor based on rolling circle amplification (RCA)-bridged two-stage exonuclease Ⅲ (Exo Ⅲ)-aided recycling amplification (Exo Ⅲ-RCA-Exo Ⅲ) was developed for highly sensitive detection of micro RNA. The analysis of DNA is accomplished by recognizing the target to a hairpin DNA that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of hairpin DNA in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA reaction with a circle DNA template. The RCA products containing thousands of repeated sequences that hybridize with TaqMan probe and then detected by Exo Ⅲ-assisted recycling amplification, After hybridization, the TaqMan probes are digested from the recessed 3’hydroxyltermini by Exo Ⅲ, releasing the fluorophores and generating enhanced fluorescence signals. Meanwhile, the RCA products with 3’ protruding ends are liberated and hybridize with another TaqMan probes, triggering another cycles and obtaining significantly increasing fluorescence. The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.32 aM.(3) In chapter 4, on the basis of a dual-amplification fluorescent sensing platform composed by rolling circle amplification (RCA) and exonuclease Ⅲ-aided recycling amplification, detections of ATP and K~+ were accomplished by taking advantage of their inhibiting effect of S1 nuclease. S1 nuclease is a single-stranded-specific endonuclease, the single-stranded RCA primer (t-DNA) would be hydrolyzed into mononucleotides due to the presence of S1 nuclease, which could not cause the RCA reaction and subsequent Exo Ill-aided recycling amplification reaction, resulting in extremely weak fluorescence. In the presence of ATP or K~+, they can protect t-DNA from the hydrolysis of S1 nuclease, which succeeds in producing RCA products and Exo Ⅲ-assisted amplification of fluorescence signals. The turn-on sensor was established, in which the presence of S1 nuclease can help to reduce the background signal, thereby getting an increased sensitivity. ATP and K~+ can be detected in a range of 5-200 nM and 0.5-5 mM with a detection limit of 0.5 nM and 2 nM respectively. In addition, this approach can also be applied for inhibitor screening of endonuclease.