Email updates

Keep up to date with the latest news and content from BMC Chemical Biology and BioMed Central.

Open Access Research article

Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

Philip Hendry1*, Maxine J McCall1, Tom S Stewart2 and Trevor J Lockett1

Author Affiliations

1 CSIRO Molecular Science, PO Box 184 North Ryde NSW 1670, Australia

2 School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney NSW 2052, Australia

For all author emails, please log on.

BMC Chemical Biology 2004, 4:1  doi:10.1186/1472-6769-4-1

Published: 9 December 2004

Abstract

Background

Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues.

Results

A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent.

Conclusion

Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.