title: NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection.
abstract: As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.
authors.affiliation: Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA.
authors.forename: Sissel
authors.initials: S
authors.lastname: Juul
authors.forename: Judy M
authors.initials: JM
authors.lastname: Obliosca
authors.forename: Cong
authors.initials: C
authors.lastname: Liu
authors.forename: Yen-Liang
authors.initials: YL
authors.lastname: Liu
authors.forename: Yu-An
authors.initials: YA
authors.lastname: Chen
authors.forename: Darren M
authors.initials: DM
authors.lastname: Imphean
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
authors.forename: Hsin-Chih
authors.initials: HC
authors.lastname: Yeh
mesh_terms: D004261:DNA Replication / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter / Q000235:genetics; D053768:Metal Nanoparticles / Q000737:chemistry*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D015335:Molecular Probes / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D004264:DNA Topoisomerases, Type I