Rapid and Label-Free Monitoring of Exonuclease III-Assisted Target Recycling Amplification

Abstract

Target recycling-oriented amplification has been widely applied for sensitive detection of DNA, RNA, and proteins due to its successful overcoming the inherent limitation of target-to-signal ratio of 1:1 in the traditional hybridization assay. Exonuclease III (Exo III) is usually used as the cleavage enzyme in the target recycling-oriented amplification because of its easy availability, high catalytic activity, and wide applicability. Even though Exo III is assumed to be double-stranded DNA (dsDNA) specific exonuclease in most literature, its cleavage of single-strand DNA (ssDNA) does occur, resulting in the target-independent degradation of probes. Herein, we design an intramolecular displacement probe with the capability of resistance to the nonspecific digestion of Exo III and fast hybridization kinetics. Through the substitution of 2-aminopurine for adenine in the intramolecular displacement probes, we develop a rapid and label-free approach to monitor Exo III-assisted target recycling amplification. We further demonstrate that this method can be used for the detection of DNA and proteins with excellent specificity and high sensitivity. Importantly, this method can be extended to rapid, label-free and multiplexed detection of various nucleic acids, proteins, and small molecules using different kinds of fluorescent nucleotide analogues and specific aptamers.

Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe

“…One dsDNA, which is obtained by hybridization between ssDNA1 and ssDNA2, acts as the substrate of this enzyme reaction. Exo III catalyzes the removal of mononucleotides from the 3 0 -terminus of dsDNA [20]. SG is an asymmetrical cyanine dye, and the fluorescence of free SG is very weak.…”

“…Escherichia coli exonuclease III (Exo III) is the major apurinic/apyrimidinic DNA-repair nuclease [19], and it has a double strand-specific and nonprocessive 3 0 ? 5 0 exodeoxyrebonuclease activity [10,20]. We used Exo III as a model enzyme to demonstrate the feasibility of our method.…”

Label-free fluorescence dual-amplified detection of adenosine based on exonuclease III-assisted DNA cycling and hybridization chain reaction

“…Exo III-assisted DNA cycling method is one of the commonly used strategies. Differing from nicking endonucleases, Exo III does not need a specific recognition site and can selectively catalyze the stepwise hydrolysis of mononucleotides from the recessed or blunt 3′-hydroxyl termini of duplex DNA (Xu et al, 2012;Gao and Li 2014;Liu et al, 2013;Zhang et al, 2013). Through Exo III-assisted cycling reaction, a large amount of single-stranded DNAs can be acquired for signal amplification.…”

Terminal Protection of Small Molecule-Linked DNA for Small Molecule–Protein Interaction Assays

“…Based on this finding, Jiang et al as well as researchers from other groups have developed a series of methods for sensitive and specific detection of the interactions between proteins and small molecules [17]. The advantage of using terminal protection assay is that it translates the binding of small molecules to proteins into the presence of a specific DNA sequence, therefore enabling the detection of small molecule–protein interaction using various DNA sequence amplification and detection technologies [18–20]. …”