Single-base mismatch discrimination by T7 exonuclease with target cyclic amplification detection.

T7 exonuclease is reported for the first time to have high specificity in discriminating single-base mismatch and utilized for developing a target cyclic amplification biosensor strategy for sensitive SNP detection based on graphene oxide quenching of uncleaved probes.

Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection.

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.

Highly sensitive and selective strategy for microRNA detection based on WS2 nanosheet mediated fluorescence quenching and duplex-specific nuclease signal amplification.

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL(-1) with a detection limit of 0.5 ng mL(-1). By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.

Graphene oxide-hairpin probe nanocomposite as a homogeneous assay platform for DNA base excision repair screening.

Uracil-DNA glycosylase (UDG) as one of the most important base excision repair enzymes plays a crucial role in protecting the genome from endogenous DNA damage and sustaining the genome integrity. Quantitative activity analysis of UDG is a central challenge and of fundamental importance in bioanalysis. Here, we proposed a novel biosensor constituted by adsorbing a fluorophore-labeled hairpin probe onto the surface of graphene oxide (GO) as a homogeneous assay platform for sensitive UDG activity assay. Active UDG could excise the uracil base in the hairpin probe, and further hydrolysis of the leaving abasic site gave rise to high fluorescence. Thus, it provided a convenient approach for UDG activity quantification. Because of the unique ability of GO in universal fluorescence quenching, a low background fluorescence signal can be obtained for the efficient fluorescence resonant energy transfer from the fluorophore-labeled on the hairpin probe to GO sheet. A quite wide dynamic range from 0.0017 U/mL to 0.8 U/mL was achieved for UDG assay and the detection limit was estimated to be 0.0008 U/mL. The results indicated that this strategy offers a simple, cost-effective, highly sensitive and selective homogeneous detection platform for UDG activity assay related biochemical studies.

Enzyme-catalyzed assembly of gold nanoparticles for visualized screening of DNA base excision repair.

Activity screening of DNA base excision repair (BER) enzymes is a crucial step for understanding numerous fundamental biochemical processes. A novel label-free homogeneous technique is developed for visualized uracil-DNA glycosylase (UDG) activity assay using gold nanoparticles (AuNPs). This strategy relies on the enzyme-catalyzed assembly of AuNPs decorated with DNA probes. In the presence of endonuclease IV (an enzyme which can further hydrolyze the products from UDG-catalyzed reaction), the substrate DNA selectively interacts with UDG followed by the efficient release of a single-strand probe. The released single-strand probe then makes the network-like assembly of decorated AuNPs to provide a visible signal for UDG activity. This strategy that can be performed in a label-free homogeneous assay format improved the duration, the simplicity and the throughput of UDG activity screening. The results provided in the present study revealed that this strategy could hold great potential as a robust, convenient and visualized platform for activity screening of uracil-DNA glycosylases with high selectivity and desirable sensitivity.