Determination of RNase H activity via real-time monitoring of target-triggered rolling circle amplification.

Abstract

The authors describe a method for real-time monitoring of the activity of ribonuclease H (RNase H). It is based on target-triggered rolling circle amplification (RCA). It utilizes a specially designed primer that contains a RNA sequence in the center and an amino group at the 3'-end. In the absence of RNase H, the primerwhen hybridized to a circular DNA template is not extended by DNA polymerase due to the amino group at the 3'-end. In contrast, the presence of RNase H specifically degrades the RNA sequence of the primer hybridized to the circular DNA template. This results in the conversion of the 3'-amino group to a 3'-hydroxy group and thereby enables the extension reaction promoted by DNA polymerase. This, consequently, leads to efficient RCA producing a long concatenated DNA strand. Its generation can be monitored in real-time by using the fluorescent dye SYBR green II which is specific for single-stranded DNA. Based on this RNase H-triggered RCA, RNase H activity can be selectively determined at levels as low as 0.019U·mL-1 with a total assay time of <5min. The diagnostic capability of this assay was demonstrated by monitoring the activity of RNase H in tumor cells. Graphical abstract Schematic of real-time monitoring of ribonuclease H (RNase H) activity based on target-triggered rolling circle amplification (RCA). RNase H that degrades RNA in primer, converts 3'-amino group to 3'-hydroxy group, which promotes RCA with fluorescence enhancement of the probe SYBR green II.