Highly Sensitive Nuclease Assays Based on Chemically Modified DNA or RNA

Abstract

Nucleolytic enzymes are associated with various diseases, and several methods have been developed for their detection. DNase expression is modulated in such diseases as acute myocardial infarction, transient myocardial ischemia, oral cancer, stomach cancer, and malignant lymphoma, and DNase I is used in cystic fibroma therapy. RNase is used to treat mesothelial cancer because of its antiproliferative, cytotoxic, and antineoplastic activities. Angiogenin, an angiogenic factor, is a member of the RNase A family. Angiogenin inhibitors are being developed as anticancer drugs. In this review, we describe fluorometric and electrochemical techniques for detecting DNase and RNase in disease. Oligonucleotides having fluorescence resonance energy transfer (FRET)-causing chromophores are non-fluorescent by themselves, yet become fluorescent upon cleavage by DNase or RNase. These oligonucleotides serve as a powerful tool to detect activities of these enzymes and provide a basis for drug discovery. In electrochemical techniques, ferrocenyl oligonucleotides with or without a ribonucleoside unit are used for the detection of RNase or DNase. This technique has been used to monitor blood or serum samples in several diseases associated with DNase and RNase and is unaffected by interferents in these sample types.

Ribonuclease-Responsive DNA Nanoswitches

“…Early methods developed to determine RNase activity include renaturation gel assays, 24 high-performance liquid chromatography (HPLC), 25 colorimetry, 26 and fluorometry. 27 These methods suffer from limitations such as complexity, high cost, and low sensitivity, 28 spurring recent detection approaches using catalytic hairpin assembly, 27 gold nanoparticle conjugates, 29 magnetic nanoparticles, 30 and DNA walkers. 31 These assays have higher sensitivity but include multiple wash steps, additional amplification, indirect quantitation, and specific equipment for readout.…”

Fluorometric Measurement of Adenosine 5'-Triphosphate Using Exonuclease V Activity

“…The method of fluorometric ATP detection using exonuclease V and DNA probes with high specificity and high sensitivity was thus developed. It is considered that this method can be applied to ATP sensing on microdevices in combination with technologies for highly sensitive nuclease assay using DNA probes [31].…”

Biomimetic Receptors and Sensors

“…The deoxyribonuclease (DNase) enzyme catalyzes hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA [ 10 ]. A variety of deoxyribonucleases are known with different specificities and biological functions.…”