Homogeneous Detection of Single Rolling Circle Replication Products

We describe a simple and straightforward approach for homogeneous and isothermal detection of individual rolling circle replication (RCR) products, which represent individual padlock probe circularization events. The RCR products constitute tens of kilobases long single-stranded tandem repeated copies of the probe sequence, and in solution, they fold into micrometer-sized random coils. The method is based on the local enrichment of fluorescence-labeled probes that hybridize to the coiled RCR products compared to the concentration of free probes in solution. We present a detailed characterization of the fluorescence-labeled products using a highly sensitive and fast microscopy setup. At a 10⁴-fold excess of free label, we were able to detect and follow individual RCR products at a signal-to-background noise ratio of 27. This high signal-to-background noise ratio leaves room for analysis in a simple detection device at higher speeds or at lower labeling ratios.