NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection

• Published in: Nanoscale Vol. 7; no. 18; pp. 8332 - 8337
• Main Authors: Juul, Sissel, Obliosca, Judy M, Liu, Cong, Liu, Yen-Liang, Chen, Yu-An, Imphean, Darren M, Knudsen, Birgitta R, Ho, Yi-Ping, Leong, Kam W, Yeh, Hsin-Chih
• Format: Journal Article
• Language: English
• Published: England 14.05.2015
• Subjects: Assaying; Beacons; Diagnostic systems; DNA Replication - genetics; DNA Topoisomerases, Type I - analysis; DNA Topoisomerases, Type I - chemistry; Enzyme activity; Equipment Design; Equipment Failure Analysis; Fluorescent Dyes - chemistry; Genes, Reporter - genetics; Human; Metal Nanoparticles - chemistry; Molecular Probe Techniques - instrumentation; Molecular Probes - chemistry; Nanostructure; Nucleic Acid Amplification Techniques - instrumentation; Parasites; Purification; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence - methods
• ISSN: 2040-3364; 2040-3372
• DOI: 10.1039/c5nr01705j

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.