title: Amplified and multiplexed detection of DNA using the dendritic rolling circle amplified synthesis of DNAzyme reporter units.
abstract: The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg(2+)-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg(2+)-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg(2+)-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg(2+)-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals.
authors.affiliation: Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem , Jerusalem 91904, Israel.
authors.forename: Fuan
authors.initials: F
authors.lastname: Wang
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Lu
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Liu
authors.forename: Lina
authors.initials: L
authors.lastname: Freage
authors.forename: Itamar
authors.initials: I
authors.lastname: Willner
mesh_terms: D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis*; D021881:DNA, Catalytic / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D054856:G-Quadruplexes; D006427:Hemin / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D008163:Luminescent Measurements; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D021881:DNA, Catalytic; D016159:Tumor Suppressor Protein p53; D006427:Hemin; D004247:DNA; D006735:Horseradish Peroxidase