ssRNA Cyclase is a thermostable ligase; the circular RNA synthesis kit can catalyze intramolecular ligation (i.e., circularization) of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) substrates. For the circularization reaction, the substrate single-stranded DNA/RNA is required to possess a 5’-phosphate group and a 3’-OH group.
ssRNA Cyclase can produce single-stranded DNA templates for rolling circle replication or rolling circle transcription experiments and next-generation sequencing, as well as generate circular RNAs of 15–500 n
Case Analysis
1. GSPure® Circular RNA Synthesis Kit (Gisai) Preparation of Circular RNA Test
Figure1. Representative result of circular RNA prepared using the GSPure® Circular RNA Synthesis Kit (Gisai). The reaction volume was 20 µL, with single-stranded RNA of 307 nt at a final concentration of 1 μM. The reaction was incubated at 45°C for 60 min, after which samples were analyzed by 6% urea denaturing polyacrylamide gel electrophoresis and fluorescence imaging following staining with SYBR™ Gold nucleic acid dye. As shown, this kit can convert over 85% of 5′-phosphorylated, 3′-hydroxyl single-stranded RNA into circular RNA within 60 min.
Q & A
1. Which molecules can this kit catalyze to undergo circularization?
GSPure® Circular RNA Synthesis Kit is a thermostable ligase that catalyzes intramolecular ligation (i.e., circularization) of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) substrates that bear a 5′-monophosphate and a 3′-hydroxyl.
