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

Philip Hendry,Maxine J Mccall,Tom S Stewart,Trevor J Lockett

doi:10.1186/1472-6769-4-1

Philip Hendry, Maxine J Mccall + Show 2 more

Open Access

https://doi.org/10.1186/1472-6769-4-1

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Abstract

BackgroundHammerhead 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.ResultsA 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.ConclusionHammerhead 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.

Highlights

Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs
A number of ribozyme designs were tested for their ability to cleave this RNA sequence either as a short (30-mer) oligonucleotide or in a transcribed RNA segment (550 nt)
These designs included standard hammerhead ribozymes (Rz) which are defined as those with a helix II consisting of four base pairs closed at the end with a four-nucleotide loop, minizymes (Mz) [17] which lack helix II and instead the two segments of conserved nucleotides are linked between A9 and G12 with a non-base-paired linker, and miniribozymes (Mrz) [19] which have a single G-C base pair replacing helix II

Summary

Introduction

Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs . As such they have potential as laboratory reagents, diagnostics and therapeutics. Hammerhead ribozymes were discovered as self-cleaving motifs in a number of small, circular, pathogenic RNAs in plants [1,2,3]. Haseloff and Gerlach [5] divided the hammerhead into a form in which the majority of the conserved nucleotides were located on the enzyme strand, with the only sequence requirements for the substrate being UH (H = A, U or C) [6,7,8]. SFcigheumreat1ic representation of Mrz-12/12-A bound to substrate S-30. Schematic representation of Mrz-12/12-A bound to substrate S-30. Nucleotides which have been further modified in this study are shown in blue

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