Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events

Abstract

DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples.

Rolling circle RNA synthesis catalyzed by RNA

“…In biology, both viroids and Hepatitis D virus (HDV) replication proceeds through RCS on circular RNA genomes mediated by proteinaceous RNA polymerases, but RCS has also been reported for circular DNA templates and proteinaceous DNA polymerases in nature ( Wawrzyniak et al, 2017 ) and in biotechnology ( Daubendiek et al, 1995 ; Givskov et al, 2016 ; Kristoffersen, 2017 ; Mohsen and Kool, 2016 ). dsDNA persistence length is somewhat shorter than dsRNA (dsDNA: 45–50 nm [140–50 nt] vs. dsRNA 60 nm [200 nt]) and stacking interactions in dsDNA are weaker than in dsRNA ( Kebbekus et al, 1995 ; Svozil et al, 2010 ).…”

Rolling Circle RNA Synthesis Catalysed by RNA

“…In biology, both viroids and Hepatitis D virus (HDV) replication proceeds through RCS on circular RNA genomes mediated by proteinaceous RNA polymerases but RCS has also been reported for circular DNA templates and proteinaceous DNA polymerases in nature (Wawrzyniak et al, 2017) and in biotechnology (Daubendiek et al, 1995; Givskov et al, 2016; Kristoffersen et al, 2017; Mohsen and Kool, 2016). dsDNA persistence length is somewhat shorter than dsRNA (dsDNA: 45-50 nm (140-50 nt) vs. dsRNA 60 nm (200 nt) and stacking interactions weaker than in dsRNA (Kebbekus et al, 1995; Svozil et al, 2010), therefore dsDNA may more readily adopt a circular shape or kinks to alleviate build-up of strain or to adopt strong bends (Wolters and Wittig, 1989), we would nevertheless expect the a similar strand displacement effect would play part.…”

Mechanically Interlocked Molecules for Biomedical Applications

“…Ho, Knudsen and co‐workers reported an analytical method for the detection of topoisomerase II (topo II) activity based on DNA‐catenanes [55] . Topo II is an important target for cancer chemotherapy.…”