aptazymes as genetic regulatory switches

Background: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially selfsplicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo. Results: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules. Conclusion: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies. Background Ribozymes whose activities are modulated by effector molecules have previously been engineered. For example, Herschlag and co-workers have previously shown that a nucleobase could complement an abasic site within the hammerhead ribozyme, restoring cleavage activity [1]. Similarly, Porta and Lizardi engineered a hammerhead ribozyme that assumed an inactive secondary structure; upon the addition of an oligonucleotide that disrupted the engineered secondary structure, the ribozyme was activated [2]. More recently, it has previously been shown that many selected nucleic acid binding species (aptamers) undergo conformational changes upon ligand binding [3–6] In addition, when aptamers are conjoined to ribozymes, the ligand-induced conformational changes in the aptamers frequently modulate the structures and hence the activities of the ribozymes [7–9] For example, binding of theophylline to the anti-theophylline aptamer [10] modulates the conformation of residue [11,12] and the overall stability of the aptamer [10,13]. Appending the anti-theophylline aptamer to the hammerhead ribozyme allowed cleavage activity to be modulated by 110-fold by Published: 4 December 2002 BMC Biotechnology 2002, 2:21 Received: 13 June 2002 Accepted: 4 December 2002 This article is available from: http://www.biomedcentral. com/1472-6750/2/21 © 2002 Thompson et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Page 1 of 12 (page number not for citation purposes) BMC Biotechnology 2002, 2 http://www.biomedcentral. com/1472-6750/2/21 theophylline [13]. Similarly, appending the anti-theophylline aptamer to a small ribozyme ligase (L1) generated an aptazyme that was activated 1,600-fold by saturating theophylline concentrations [14]. The methods for engineering and selecting Group I aptazymes appear to be generalizable to several different types of ribozymes. Adjoining aptamers to the Group I self-splicing intron would potentially allow the control of splicing not only in vitro, but in vivo. To this end, we have attempted to generate and assay Group I aptazymes. Results and Discussion Design of Group I aptazymes In order to convert the Group I self-splicing intron to a regulatable aptazyme, it was necessary to first identify sequences or structures in the ribozyme whose conformation might modulate splicing activity. The structural and kinetic properties of the Group I self-splicing intron from the thymidylate synthase (td) gene in bacteriophage T4 have been extensively studied. In particular, a series of nested deletions of the P6 stem-loop have been shown to partially or completely compromise ribozyme activity [15]. More importantly, either magnesium or the tyrosyl tRNA synthetase from Neurospora mitochondria (CYT18) can suppress many of these defects [16,17]. Previous studies with other introns have also revealed that deletion of the P5 stem-loop can modulate ribozyme activity [18,19]. We hypothesized that sites where deletions modulated ribozyme activity might also prove to be sites where aptamer conformational changes would modulate ribozyme activity. Based on these studies, we designed a series of Group I aptazymes in which the anti-theophylline aptamer was substituted for either a portion of P6 or P5 (Figure 1). The anti-theophylline aptamer was attached at the same point as had previously proven successful in the design of theophylline-dependent cleavases and ligases [13,14]. Activation in vitro The self-splicing activities of the Group I aptazymes were examined in vitro using a standard splicing assay (Table 1). Magnesium had previously been shown to suppress structural defects in the td intron, and ligand-induced suppressions of splicing defects were examined by carrying out splicing reactions at either low (3 mM, stringent) or high (8 mM, permissive) magnesium concentrations [15]. Several of the constructs were either relatively inactive (e.g., Th3P6, Th5P6, and Th6P6) or did not consistently show differential splicing activity (e.g., Th4P6 and Th2P5). However, four of the constructs, Th1P6, Th2P6, Th3P6, and Th1P5, showed good activity (>10% of wild-type) and increased self-splicing in the presence of theophylline. For example, Th2P6 showed the greatest activation (8-fold at 8 mM MgCl2), while Th1P5 showed low activation (1.2-fold) at both magnesium concentrations. For all of the aptazymes except Th3P6, the ligand-induced splicing activity was greater in a standard assay at the more stringent (3 mM) magnesium concentration (Table 1). The construct Th3P6 was inactive at lower magnesium concentrations, but underwent ligand-induced splicing at the more permissive concentration (8 mM). Interestingly, those aptazyme constructs that showed ligand-dependent activity closely resembled the original deletion variants that showed magnesium-dependent recovery of splicing activity. For example, the junction between the aptamer and the Group I ribozyme in the aptazyme Th2P6 resembled the deletion variant td ∆P6-6; the aptazyme was activated by theophylline, while the splicing defect of the deletion variant at 3 mM magnesium was suppressed by 8 mM magnesium or by stabilization of a tetraloop sequence that capped the P6 helix [15]. Defects that poise a ribozyme between active and inactive conformers have previously been used to engineer effector-dependence [20]. The kinetics of splicing of two of the most activated variants, Th1P5 and Th2P6, were examined in the presence and absence of theophylline (Figure 2). As in the single point assays, the theophylline-dependence of the engineered introns was readily apparent (Figure 2a). The time courses reveal that the extents of splicing of the aptazymes were significantly affected by the addition of theophylline (Figures 2b,2c). The aptazyme modified at P5 (Th1P5) showed less theophylline-dependent enhancement of the extent of reaction than the aptazyme modified at P6 (Th2P6), consistent with the single point assays described above. Th1P5 was activated 1.4-fold at 15 minutes (Figure 2b), while Th2P6 showed a 4.0-fold rate enhancement at Table 1: Relative in vitro splicing activity of constructs containing anti-theophylline aptamers. Extent of reaction is relative to the parental construct in 3 mM MgCl2 with no theophylline at 2 hrs.