An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, 
and tested for self-splicing activity. Precursor RNAs from these constructs 
spliced efficiently and produced circular exons in vitro. Using group I 
permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA 
component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding 
element were generated and tested for activity and stability. The activity of 
circular ribozymes is comparable to the linear counterparts with similar core 
sequences. Circular forms of the TAR and Rev-binding element showed specific 
binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential 
for using this methodology to express circular RNA in vivo, circular forms of 
the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA 
indicated that the precursor RNA spliced efficiently and accurately to produce 
circular ribozymes. The activity of in vivo expressed circular ribozymes could 
be demonstrated indicating that they fold into active conformation. These 
results suggest that self-splicing group I PIE sequences could prove useful for 
expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in 
cells.