Development of a new on/off adaptor version for HDV ribozyme

Initially, a new design that considers the blocker and biosensor domains as distinct entities was performed by including them at each end of the P2 stem of the HDV ribozyme in a process that involved several steps (Figure 1B). First, a blocker sequence that base-pairs with the binding domain of the ribozyme was added to the 5′-end. The resulting ribozyme exhibited no cleavage activity with a model substrate derived from the internal ribosome entry site (IRES) region of the hepatitis C virus (HCV) (Figure 1C, lane 3). The blocker acts as a “safety lock” placing the ribozyme in an inactive conformation (off state). When the ribozyme possesses a 12 nucleotide (nt) biosensor sequence complementary to the substrate at its 3′-end, it exhibited an improved cleavage activity (Figure 1C, lane 4). The presence of an inappropriate biosensor domain (i.e. a sequence not complementary to the substrate) resulted in a cleavage level comparable to that of the original ribozyme (Figure 1C, lane 5). When an appropriate biosensor was added to a ribozyme that already possessed a blocker, a dramatic increase in the cleavage activity, as compared to that observed in the presence of an inappropriate biosensor, was observed (Figure 1C, lanes 6 and 7, respectively). Thus, the ribozyme can be switched off and on by independent domains in a way reminiscent of the MAP kinases.

Kinetic analyses were performed in order to determine the second order rate constants (k_cat/K_M') as an indication of the substrate specificities. The HDV on (i.e. the one including both a blocker and an appropriate biosensor) exhibited a cleavage activity characterized by a relative k_cat/K_M' 6-fold higher than that of the original ribozyme, which in turn had a value 12-fold higher than that of the HDV off ribozyme (i.e the one including a blocker and an inappropriate biosensor) (see Table 1). The difference between the k_cat/K_M' values of the off and on ribozymes is therefore greater than 60 times. This difference is smaller than the determined 1500 to 2500 times observed between the on and off forms of the original configuration of the SOFA module on other HDV ribozymes [6], [7]. The larger difference in the latter case is likely due to the fact that its configuration is probably better adapted to the architecture of this particular ribozyme.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Kinetic parameters of four cleaving nucleic acid enzymes in single turnover conditions

https://doi.org/10.1371/journal.pone.0000673.t001

As the specificity of a ribozyme is commonly defined as its ability to discriminate between similar substrates [1], a collection of five substrates possessing identical ribozyme binding domains, but different biosensor sequences, was synthesized (Figure 1D). Because these substrates harbor different 5′-ends, they can be distinguished by their electrophoretic migration (Figure 1D, lane 1). When the 5 substrates were incubated together with only one HDV on ribozyme, solely the substrate possessing the sequence perfectly complementary to the biosensor was cleaved (Figure 1D, lanes 2-6). Conversely, an original ribozyme (i.e. one without both a biosensor and a blocker) had the ability to cleave all of the substrates, although at different levels (Figure 1D, lane 7). Together, these data demonstrate the improvement, in terms of substrate specificity, resulting from the addition of both a blocker and a biosensor. Moreover, it shows that a ribozyme, when activated by the proper substrate, does not cleave other substrates via a trans-cleavage mechanism. Finally, the potential to cleave HCV-derived transcripts was tested in cell culture (data not shown). Only small amounts of transcripts were cleaved. Determination of the half-life of the new conformation of the SOFA-HDV ribozymes according to a procedure reported previously [8], revealed a relatively low molecular stability of these ribozymes (a likely by-product of the single-stranded extremities that these ribozymes possess). More important is that it has been possible to adapt the target-dependent module of activation from the MAP kinase to the HDV ribozyme.

Adaptation of this mode of activation to various cleaving nucleic acid enzymes

Subsequently, we tried to adapt this mode of activation to other catalytic nucleic acids. As a first attempt, we chose three cleaving molecules: the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme (Figure 2) [9]–[11]. All of these nucleic acid enzymes share a relatively similar architecture. The cleavage site of the substrates is located in the middle of the two stems formed between the catalytic center. The sequences of both of these ribozymes and of the deoxyribozyme have been reported to cleave various substrates that were used [12]–[15]. Each of these catalytic nucleic acids showed significant cleavage of their 5′-end labelled substrates, although at different levels (Figure 2A–C, lane 2). The addition of a blocker domain that “zips off” the binding domain dramatically decreased the cleavage activity (i.e. Figure 2A–C, lane 3). As is observed with the biosensor-blocker HDV ribozyme, the specificity of these engineered nucleic acid molecules was dramatically increased through blocker-mediated inactivation followed by reactivation via an extended complementarity with the appropriate biosensor sequence (Figure 2A–C, lanes 4 to 7). With both the biosensor and blocker domains, the presence of an inappropriate biosensor locked the catalytic core into an off state, while the presence of an appropriate biosensor permitted the switch into the active mode, by displacing the blocker, solely in presence of its specific substrate. The biosensors interact through more base pairs than do the blockers, thereby favoring the on state over the off state. When comparing the k_cat/K_M' values for the off and on versions of these catalytic molecules, the overall improvements were of a minimum of three orders of magnitude (Table 1). Specifically, the k_cat/K_M' values vary from 2588-, 2762- and 9167-folds between the off and on version of hammerhead ribozyme, hairpin ribozyme and 10-23 deoxyribozyme, respectively. This illustrates the important gain in terms of specificity. These important differences were mainly the result of dramatic decreases in the k_cat values between those of the original structures and those of their respective off states, suggesting that they lost their abilities to perform non-specific cleavage. Importantly, the addition of both a blocker and a biosensor permits the generation of cleaving nucleic acid enzymes that operate in a target-dependent fashion.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Autoradiograms of cleavage assays performed with controlled cleaving nucleic acid enzymes.

(A) (B) and (C) are the reactions performed with the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme, respectively. In each case the structures of the nucleic acid enzyme with the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels, and the control (-) was performed in the absence of any nucleic acid enzyme (lane 1), and, lane 2, in the presence of the unmodified version of the ribozyme. Lane 3 is the version extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate. Finally, lanes 6 and 7 are the on and off versions, respectively. The nucleotide sequences of each nucleic acid enzyme are depicted in the Figures S3pone.0000673.s004 to S5.

https://doi.org/10.1371/journal.pone.0000673.g002

Engineering ribozymes catalyzing various reactions

Next, we investigated other ribozyme's catalyses. As a first attempt, the ligation catalyzed by the hairpin ribozyme was studied. Extending the 3′-end of the original hairpin ribozyme by 6 nt resulted in the retention of the cleavage product, and consequently favored the reverse reaction (Figure 3A) [14]. Using a 5′-³²P-labelled RNA strand possessing a terminal 2′-3′-cyclic phosphate and a second RNA strand possessing a 5′-hydroxyl resulted in the detection of ligation products (Figure 3B). The addition of a biosensor with a sequence complementary to that of the substrate increased the amount of ligation observed. In contrast, the addition of the blocker module almost completely abolished the ligation. Finally, an on version, which includes both the blocker and an appropriate biosensor, exhibited a ligation activity of up to 65%; while an off version (i.e. one with an inappropriate biosensor) showed less than 5% activity.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Catalytic activity of the engineered dependent ligating and capping ribozymes.

(A) and (C) are the nucleotide sequences and secondary structures of the ribozyme-substrate complexes of the ligating hairpin and capping ribozymes, respectively. (B) and (D) are autoradiograms of the PAGE gels used to analyse the ligation and capping reactions, respectively. The schematic structures of the nucleic acid enzymes with both the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels. The controls (-) was performed in the absence of ribozyme (lane 1), and, lane 2, in the presence of the unmodified versions. Lane 3 is the ribozyme extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate, respectively. Finally, lanes 6 and 7 are the on and off ribozymes, respectively. The nucleotide sequences of each engineered nucleic acid enzyme are depicted in Figures S6 and S7.

https://doi.org/10.1371/journal.pone.0000673.g003

Subsequently, the concept was adapted to an artificial ribozyme that catalyzed the attack of a 5′-terminal nucleotide phosphate on the α-phosphate of the terminal triphosphate of an RNA substrate, forming a 5′-5′ phosphodiester linkage (i.e. a capping ribozyme; Figure 3C–D) [16]. A version harboring a blocker alone exhibited only residual activity for the capping of a 33-nucleotide substrate (<3%), while one possessing only an appropriate biosensor sequence was slightly more efficient than the original. The on version has the ability to switch from its off to on state, while the off version remained trapped in the inactive conformation.

DNA templates and constructs

Deoxyribozymes were purchased as oligonucleotides from Invitrogen. All ribozymes and small RNA substrates used were produced by PCR using oligonucleotides purchased from either Invitrogen or IDT Inc. Briefly, DNA oligonucleotides corresponding to the sequences complementary to the different templates, and possessing a T7 promoter complementary sequence, were annealed to T7 promoter primer and the primers extended in PCR reactions. The reaction products were then used as templates for run-off transcription (see below). Alternatively, some constructs were prepared by annealing of two overlapping oligonucleotides that were then filled in by PCR using Vent DNA polymerase (New England Biolab), producing a double-stranded DNA fragment. Each of these fragments was provided with a T7 promoter at the beginning of the sense strand for subsequent use as a template for run-off transcription. Hepatitis C virus (HCV) derived substrate (a 575 nt long template containing the internal ribosome entry site (IRES) and the first part of the ORF) was produced by KpnI digestion of the pHCVA plasmid [6], which contains 1348 nt of the 5′ sequence of hepatitis C virus (genotype 1b) [24]. PCR products and linearized plasmid templates were purified by phenol/chloroform extraction, precipitated with ethanol and dissolved in water.

RNA synthesis

Run-off transcriptions were performed as described previously [6]. Briefly, transcriptions were performed using purified T7 RNA polymerase (10 µg) in the presence of RNA Guard (24 U, Amersham Biosciences) and pyrophosphatase (0.01 U, Roche Diagnostics) in a buffer containing 80 mM HEPES-KOH, pH 7.5, 24 mM MgCl₂, 2 mM spermidine, 40 mM DTT and either linearized plasmid DNA (5 µg) or PCR product (100 pmol) as template. Each NTP (5 mM) was added to the mixture either with or without 50 µCi of [α-³²P]UTP (3 000 Ci/mmol, New England Nuclear) in a final volume of 100 µL, and the reaction incubated at 37°C for 2 to 4 h. Upon completion, the reaction mixtures were treated with DNase RQ1 (Promega) at 37°C for 20 min, and the RNA then purified by phenol:chloroform extraction and precipitation with ethanol. RNA products were fractionated by a denaturing (7 M urea) 6 to 20% polyacrylamide gel electrophoresis (PAGE; 19∶1 ratio of acrylamide to bisacrylamide) using 45 mM Tris-borate, pH 7.5/1 mM EDTA solution as running buffer. The reaction products were visualized either by UV shadowing, or by autoradiography. The bands corresponding to the correct sizes for both the ribozymes and the substrates were excised from the gel and the transcripts eluted overnight at room temperature in a buffer containing 1 mM EDTA, 0.1% SDS and 0.5 M ammonium acetate. The transcripts were ethanol precipitated at −80°C, washed, dried and dissolved in water. The amounts retrieved were determined either by ³²P counting, or by absorbance at 260 nm.

In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences). The enzyme was inactivated by incubating at 65°C for 5 min. Dephosphorylated RNA (5 pmol) was then radiolabelled at its 5′-end using 3 U of T4 polynucleotide kinase in the presence of 3.2 pmol of [γ-³²P]ATP (6000 Ci/mmol, New England Nuclear). Radiolabelled transcripts were purified by electrophoresis on a denaturing gel and recovered as described above.

Cleavage assays and kinetics

All reactions with the cleaving HDV, hammerhead and hairpin ribozymes, as well as those with the deoxyribozyme, were performed under single-turnover conditions ([Rz]>>[S]) using 100 nM of nucleic acid enzyme and trace amounts of 5′-³²P-labelled substrates at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 7.5 and 10 mM MgCl₂. After an incubation of 1 h, the reactions were quenched by the addition of ice-cold formamide dye buffer (1 vol of 97% formamide, 10 mM EDTA, pH 8.0, 0.25% xylene cyanol, 0.25% bromophenol blue) and fractionated on 6% to 20% PAGE gels. The gels were then analyzed with a radioanalytic scanner (Storm, Molecular Dynamics). For time-course experiments, aliquots of 0.8 µL were taken at various times up to 3 h and treated as described above. Kinetic analyses were performed under single turnover conditions. Trace amounts of 5′-³²P-labelled substrate (<1 nM) were cleaved by various concentrations of either ribozyme or deoxyribozyme (5–400 nM). The fraction cleaved was determined, and the rate of cleavage (k_obs) obtained from fitting the data to the equation A_t = A_□(1-e^−kt) where A_t is the percentage of cleavage at time t, A_□ is the maximum percent cleavage (or the end-point of cleavage), and k is the rate constant (k_obs). Each rate constant was calculated from at least two independent measurements. The values of k_obs obtained were then plotted as a function of nucleic acid enzyme concentration for the determination of k_cat, K_M' and k_cat/K_M'. The values obtained from independent experiments varied by less than 15%. The experiments of substrate specificity using several substrates were performed under multiple turnover conditions using 100 nM HDV ribozyme and 1 µM substrate.

Ligation reactions

Ligation reactions were performed as described previously [14], [15] with minor modifications. Full length 5′-³²P-labelled substrate (5 pmol) was cleaved in the presence of 10 µM of hairpin ribozyme in a volume of 50 µl containing 50 mM Tris-HCl, pH 7.5 and 10 mM MgCl₂. The mixture was incubated 2 h at 37°C, quenched by the addition of ice-cold formamide dye buffer, purified on a 20% denaturing PAGE gel and the radiolabelled 20 nt 5′-product recovered as described. This RNA strand was then used as substrate for the ligation reaction. The 3′-product used in this ligation reaction was purchased from IDT Inc. This deprotected 5′-hydroxyl RNA was purified as described above. Ligation reactions were performed using 10 µM of the hairpin ribozyme in the presence of 15 µM of the 3′-end substrate and trace amounts of 5′-³²P-labelled substrate mixed in a total volume of 9 µl containing 50 mM HEPES-KOH, pH 7.0. The reactions were preheated at 37°C for 10 min so as to permit the annealing of the substrate to the ribozyme, and the ligation reactions started (time = 0) by adding 1 µL of 150 mM MgCl₂. Samples were incubated at 37°C for 1 h, fractionated on gels and visualized by autoradiography.

Capping enzyme reaction

The capping reaction was performed as described previously [16]. Substrate was 3′-end labelled by incubating, at 37°C, 10 pmol of transcripts with 3.2 pmoles of [³²P]Cp (3000 ci/mmol, New England Nuclear) and 1 U T4 RNA ligase (New England Biolab) in the buffer supplied by the manufacturer supplemented with 10% DMSO. After 3 h, the reaction was stopped and the transcripts purified by denaturing 10% denaturing PAGE and recovered as described above. Capping reactions were carried out under single turnover conditions. Both the ribozyme (2 µM) and substrate (0.2 µM) were annealed together for 5 min at 22°C in a reaction buffer containing 50 mM Tris-HCl, pH 7.5, 25 mM MgCl₂ and 150 mM KCl. The reactions were initiated by the addition of ATP to a final concentration of 1 mM. After 16 h of incubation at 22°C, the reactions were quenched by addition of ice-cold formamide dye buffer and analyzed on denaturing 15% PAGE gels.