The primary target site for cleavage in this study was a previously identified site 23 in the cps II mRNA 22 of Plasmodium falciparum. The chosen target site, centred at position 3733 in the nucleotide sequence (Genbank reference L32150), has the local sequence 5' UAA CUU AUC AAG GUC* AAG AAC AUG AUG UUC 3', where the site of cleavage is denoted by the asterisk. 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 (in this case consisting of 5 nucleotides), and finally miniribozymes (Mrz) 19 which have a single G-C base pair replacing helix II and in this instance a linker sequence consisting of four deoxythymidines. In this communication the core of each ribozyme is flanked by hybridising arms composed of DNA of various lengths, where the length is given in the ribozyme's name (e.g. Rz-6/12 has 6 nt in its 5' hybridising arm and 12 nt in its 3' arm). Hammerhead-ribozyme derivatives of these designs were tested for their ability to cleave the 30-mer substrate at pH 7.6, 37°C and 10 mM MgCl₂ under pseudo first-order conditions with an excess of ribozyme. Ribozyme concentrations ranged from 50 nM to 10 μM. Cleavage data fitted well to single exponential curves to yield observed rate constants which were plotted against the ribozyme concentration in each experiment to determine the apparent dissociation constant ("K_d") and the maximum cleavage rate constant (k_max) for each ribozyme-substrate pair (Table 1). In terms of catalytic efficiency, the most efficient ribozyme was the standard hammerhead Rz-12/12. Its efficiency is due to a very low "K_d" of 7 nM, despite displaying a k_max some 5-fold less than Mrz-12/12. The highest k_max was displayed by Rz-6/12; however it had a "K_d" about 60-fold greater than Rz-12/12.

The abilities of all the ribozymes to cleave the same target in the context of transcribed RNA (550 nt) was also examined. Mrz-12/12, by virtue of only a modest decrease in k_max, and marginal increase in "K_d", was by far the most efficient of all the designs tested. In contrast Mrz-8/8, Rz-12/12 and Rz-6/12 displayed "K_d"'s between 2 and 3 mM, which, in the case of Rz-12/12, is an increase of about 400-fold. Interestingly the k_max values displayed by Rz-12/12 and Rz-6/12 were within experimental error, and were the same as displayed by Rz-12/12 for cleavage of the short substrate.

We examined whether the effectiveness of the miniribozyme design was limited to this target site. Miniribozymes, with long (>10 nucleotides) hybridising arms were designed to cleave RNAs of interest to other projects in the laboratory. Tet Mrz was a 53-mer oligonucleotide with conserved bases of RNA and hybridising arms and stem loop II composed of DNA. This Mrz, with hybridising arms of 18 and 19 nucleotides, was targeted to cleave the GUC triplet at position 60 of the Tetrahymena IVS ribozyme 24. It cleaved the 388-nt transcript, L-21 ScaI, with a rate constant of 4.0 ± 0.2 min^-1 (t_1/2 = 10 seconds), to about 70%, at pH 7.6, 37°C and 10 mM MgCl₂. Another miniribozyme with 14-mer arms (HC Mrz), targeting a segment of Hepatitis C polyprotein mRNA, was tested against a synthetic 29-mer substrate. Under our standard conditions about 70% of the substrate was cleaved, and the cleavage rate (>5 min^-1) was too fast to be measured reliably. In contrast, the standard ribozyme HC Rz cleaved 78% of the same substrate with a rate constant of only 0.2 ± 0.02 min^-1. Finally, PDGF MRz, with hybridising arms each of 10 ribonucleotides, was tested against both 25-mer synthetic and 707-nt transcribed substrates. Under standard conditions, the 25-mer was ~ 80% cleaved with a rate constant > 5 min^-1. Reducing the magnesium ion concentration to 1 mM yielded a rate constant of 2.8 min^-1 and 63% cleavage for the 25-mer substrate, and 1.6 min^-1 and 45% cleavage for the 707-nt transcript.

Mrz-12/12 was the most efficient cleaver of the 550-nt cpsII RNA transcript and was used as the platform to test the effect of chemical modification on cleavage activity and nuclease stability. Firstly, the stability of unprotected Mrz-12/12 in RPMI + 10% human serum was determined using ³²P 5'-end labelled ribozyme at 37°C (Figure 2). Approximately 90% of the miniribozyme is degraded in 10 seconds, and no full-length material is observed after 10 minutes. Initially there are three main sites of cleavage, at U₄, U₇ and C_15.2. After 10 minutes, nearly all the end-labelled material co-migrates with the band generated by alkaline digestion at U₄. That initial, major product has a half-life of about two hours under these conditions, as it is slowly converted to a product one nucleotide shorter, ie its 3' end corresponds to C₃ in the original miniribozyme.

Commercially available modified phosphoramidites were used to generate hammerhead derivatives which were expected to be protected from nuclease degradation. The effect of the various modifications on the cleavage ability are given in Table 2. In these experiments the concentration of the miniribozyme was fixed at 1 μM and the substrate S-30 at 5 nM. The nucleotide most sensitive to chemical modification was U₄. All the 2' modifications tested (amino, deoxythymidine, deoxyuridine, and O-methyl, shown as D, F, G and H, respectively, in Table 2) diminished activity by more than 10-fold. Only the presence of a phosphorothioate linkage between U₄ and G₅ preserved the activity of the unmodified ribozyme. The ready availability of 2'-O-methyl phosphoramidites, coupled with the previous demonstrations 916 of tolerance to that modification at C₃, U₇ and C_15.2, lead us to synthesise Mrz-J, which we expected to have reasonable activity and nuclease stability. Its cleavage rate constant was actually twice that observed for the unmodified ribozyme, and its stability in serum was increased about 10³-fold. The kinetics of degradation in serum (Figure 3) were not straight-forward, displaying a rapid initial decay of about 25% of the starting material, followed by an approximately first-order decay with a half-life around 30 minutes. This second phase accounted for approximately 50% in the total starting material, ie after about 4 hours approximately 25% of the full-length material remained intact and thereafter decayed only very slowly. There was a single major product, corresponding to cleavage at U₄, observed over the six hours of the experiment.

The single degradation site in Mrz-J suggested that a more robust modification at this position would have a significant effect on its lifetime in serum. Mrz-12/12 K was synthesised with a 2'-amino modification at the U₄ position and with a 2'-O-methyl modification at each of the three other conserved pyrimidines. Protection of the 3' end was omitted from Mrz-12/12 K because it did not appear to contribute significantly to the stability observed in Mrz-12/12 J. As expected, the cleavage activity of Mrz-12/12 K was significantly reduced compared to Mrz-12/12 J (Table 2), but stability in serum was greatly improved with only minor losses apparent after 5 hours incubation at 37°C (Figure 4). Even after 24 hours in 10% serum, approximately 25% of the radioactivity co-migrated with the full-length material, representing an almost 10⁴-fold increase in stability. Even in the absence of 3' terminal protection there was no 3' exonuclease activity apparent. The amount of ³²P label appearing in the gel lanes remained relatively constant throughout the experiment implying a lack of significant phosphatase activity in the serum. The main sites for degradation were the remaining unprotected (purine) ribonucleotides.

The modifications to the conserved nucleotides in this study were all made in the context of a miniribozyme. Compared to a standard hammerhead, the catalytic domain is expected to be more conformationally flexible, and therefore it should not be assumed that all the changes described here can be directly applied to standard hammerheads with a helix II of four base-pairs. However, as for the miniribozyme, a standard ribozyme containing a 2'-amino modification at U₄, was about 15-fold less efficient at cleaving S-30, (k_obs = 0.05 min^-1, Rz-12/12-L, Table 2), compared to the unmodified ribozyme (Rz-12/12).

Oligoribonucleotides were synthesised using DNA phosphoramidites and ancillary reagents supplied by Perkin Elmer (Applied Biosystems Division, Foster City, CA), RNA and modified phosphoramidites from Glen Research (Sterling, VA) and using an Applied Biosystems Model 394 DNA synthesiser. Phosphorothioates were introduced by oxidation with Beaucage reagent (Glen Research, Sterling, VA). Deprotection and purification of oligonucleotides were as described previously 20. The purity of each oligonucleotide was checked by labelling its 5'-end with ³²P phosphate using T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) and [γ-³²P]-ATP (Du Pont, Wilmington, DE), followed by electrophoresis in a 15% polyacrylamide gel containing 7 M urea and visualisation using a Molecular Dynamics PhosphorImaging system (Sunnyvale, CA). The concentrations of the purified oligonucleotides were determined by UV spectroscopy using the following molar extinction coefficients for the various nucleotides at 260 nm: A, 15.4 × 10³; G, 11.7 × 10³; C, 7.3 × 10³; T/U, 8.8 × 10³ l mol^-1 cm^-1. All oligonucleotides were stored in distilled, deionised and autoclaved water at -20°C.

The 550-nt CPSII substrate was transcribed by T7 RNA polymerase from the vector pCPS3b.1 22 after linearisation with Eco RI restriction enzyme using an Ampliscribe T7 kit (Epicentre Technologies, Madison, WI, USA). The transcription reaction contained CTP, GTP and ATP (unlabelled) at 5 mM, UTP at 0.5 mM and ³²P UTP at about 1 nM. The transcription was for 90 minutes at 37°C, followed by the addition of 0.5 μL of DNAaseI and incubation for a further 20 minutes. The mixture was extracted three times with a 1:1 phenol/chloroform mix and once with chloroform, before precipitation of the transcript by the addition of 1/3 volume of 7.5 M ammonium acetate and incubation on ice for at least 2 hours. After recovery of the transcript by centrifugation and washing, the degree of incorporation of ³²P-labelled uridine was measured by Cerenkov counting of the transcript and the unincorporated UTP. The Tetrahymena intervening sequence transcript was generated from the ScaI digested pT7L-21 vector 24 (ATCC 40291) using the same method. The PDGF transcript was generated by T3 RNA polymerase transcription from the Eco RI digested vector pBSPDGF-LA using an Ampliscribe T3 kit (Epicentre Technologies, Madison, WI, USA). The vector pBSPDGF-LA was generated by insertion of the Xba I/Eco RI fragment from pmetPDGF-LA 42 into a similarly digested pBluescribe vector.

Deoxyribonucleotides are denoted by lower case letters, ribonucleotides by uppercase letters, modified nucleotides are shown in bold, C^f = 2'-fluoro-2'-deoxycytidine, C^me = 2' O-methylcytidine, U^am = 2'-deoxy-2'-aminouridine, U^me = 2' O-methyluridine, ps = phosphorothioate linkage. S-30, UAA CUU AUC AAG GUC* AAG AAC AUG AUG UUc, * denotes site of cleavage; Mrz-8/8, atgttctt CUGAUGA gttttc GAAAC cttgat; Mrz-12/12, catcatgttctt CUGAUGA gttttc GAAAC cttgataagt; Mz-12/12 catcatgttctt CUGAUGA gtttt GAAAC cttgataagt; Rz-12/12, catcatgttctt CUGAUGA GUCC UUUU GGAC GAAAC cttgataagt; Rz-6/12, gttctt CUGAUGA GUCC UUUU GGAC GAAAC cttgataagt; Mrz-12/12-A (= Mrz-12/12), catcatgttctt CUGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-B, catcatgttctt C^fU^amGAU^amGA gttttc GAAAC^f cttgataagt; Mrz-12/12-C, catcatgttctt CU^amGAU^amGA gttttc GAAAC cttgataagt; Mrz-12/12-D, catcatgttctt CU^amGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-E, catcatgttctt CUGAU^amGA gttttc GAAAC cttgataagt; Mrz-12/12-F, catcatgttctt CtGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-G, catcatgttctt CuGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-H, catcatgttctt CU^meGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-I, catcatgttctt CUpsGAUGA gttttc GAAAC cttgataagt; Mrz-12/12-J, catcatgttctt C^meUpsGAU^meGA gttttc GAAAC^me cttgataagtpstpst; Mrz-12/12-K, catcatgttctt C^meU^amGAU^meGA gttttc GAAAC^me cttgataagt; Rz-12/12-L, catcatgttctt CU^amGAUGA GUCC UUUU GGAC GAAAC cttgataagt; Tet Mrz, gcaatctattggtttaaa CUGAUGA gttttc GAAAC tagctaccaggtgcatg 3'; HC Mrz, 5' gtcgccacgacgac CUGAUGA gttttc GAAAC gttcccgctggt 3'; HC Rz, 5' gtcgccacgacgac CUGAUGA GGCC GAAA GGCC GAAAC gttcccgctggt 3'; HC S29, 5' ACCAGCGGGAACGUCGUCGUCGUGGCGAc 3'; PDGF Mrz, 5' CAGCUUCCUC CUGAUGA ggtaac GAAAU GCUUCUCt 3' ; PDGF S25, 5' GAAGAGAAGCAUCGAGGAAGCUGUc 3'.

Cleavage kinetics were studied at 37°C, pH 7.6 and 10 mM MgCl₂ under conditions of ribozyme excess; the substrate concentrations varied between 4 and 20 nM, and the ribozyme concentrations were between 20 nM and 10 μM. The short, synthetic substrates were labelled at their 5' ends using polynucleotide kinase (Roche Molecular Biochemicals) and γ-³²P ATP. The long, transcribed substrates were uniformly labelled by including α-³²P UTP in the transcription reaction. The ribozymes and ³²P-labelled substrates were mixed together in 20 μL of 50 mM Tris buffer and heated to 85°C for two minutes, then incubated at 37°C for 2 minutes. The tube containing the mix was centrifuged, and 2 μL was removed and put into 4 μL quenching solution which contained 90% formamide, 20 mM EDTA and 0.01 % xylene cyanol and bromophenol blue. The reaction was initiated by the addition of 2 μL of 100 mM MgCl₂, and 2 μL samples were removed at various times and quenched as described above. The reaction products were separated using 15% denaturing polyacrylamide gels (containing 7 M urea), and then imaged using a Molecular Dynamics PhosphorImager. At each time-point, the amount of the substrate cleaved was calculated and plotted versus time. The data were fitted by a least-squares method using the program MacCurveFit 43, to an equation of the form: P_t = P_∞-(exp(-k_obst)P_Δ) where P_t is the amount of product at time t, P_∞ is amount of product generated in the exponential phase of the reaction, k_obs is the first-order rate constant for the reaction, and P_Δ is the difference in the amount of product at t = 0 and P_∞. Some reactions resulted in biphasic kinetics in which the first-order phase described above was followed by a slower step which was accommodated adequately in the curve-fitting process by the addition of a linear term, P_t = {P_∞-(exp(-k_obst)P_Δ)} + d*t. Apparent dissociation constants "Kd" and the maximal first-order rate constants k_max were determined from the plot of k_obs versus ribozyme concentration according to the simple binding isotherm: k_max = (k_obs * [Rz])/("K_d" + [Rz]) by least-squares fitting using the program MacCurveFit.

Ribozymes labelled at their 5' end with ³²P phosphate were dissolved in RPMI medium (Gibco) at 2 μM and the reactions were initiated by adding human serum (pooled human serum, Red Cross Blood Bank, Sydney, NSW), to a final concentration of 10%. A 2 μL sample was removed immediately and the remainder incubated at 37°C with samples removed at the times indicated in Figures 2,3,4. The samples were quenched by addition to 4 μL of 90% formamide containing 20 mM EDTA and 0.01% bromophenol blue and xylene cyanol. The products of nuclease digestion in each sample were separated on 15% polyacrylamide gels containing 7 M urea and imaged using a Molecular Dynamics PhosphorImager.