Rational Design of Allosteric Ribozymes Jin Tang and Ronald R Breaker
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Research Paper 453 Rational design of allosteric ribozymes Jin Tang and Ronald R Breaker Background: Efficient operation of cellular processes relies on the strict Address: Department of Molecular, Cellular and control that each cell exerts over its metabolic pathways. Some protein enzymes Developmental Biology, Yale University, New are subject to allosteric regulation, in which binding sites located apart from the Haven, Connecticut 06520-8103, USA. enzyme’s active site can specifically recognize effector molecules and alter the Correspondence: Ronald R Breaker catalytic rate of the enzyme via conformational changes. Although RNA also E-mail: [email protected] performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems, It has recently been established that Key words: aptamer, ATP, RNA engineering, RNA enzyme, self-cleaving RNA small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind Received: 16 April 1997 corresponding ligand molecules. We set out to examine whether the catalytic Revisions requested: 9 May 1997 activity of an existing ribozyme could be brought under the control of an effector Revisions received: 16 May 1997 Accepted: 20 May 1997 molecule by designing conjoined aptamer-ribozyme complexes, Chemistry & Biology June 1997, 4:453-459 Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have http://biomednet.com/elecref/1074552100400453 created the first example of an allosteric ribozyme that has a catalytic rate that 0 Current Biology Ltd ISSN 1074-5521 can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach,,allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created. Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation -a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules. Introduction It then seems likely that small-molecule effecters could The regulation of metabolic processes involves a variety of perform similar functions. Indeed, small antibiotic mol- molecular mechanisms including transcriptional and trans- ecules have been shown both to inhibit and enhance the lational control, RNA and protein degradation, and cat- catalytic rates of certain ribozymes [14-161, although these alytic induction and inhibition. A number of enzymatic molecules typically bind directly to the catalytic domain of processes are subject to allosteric regulation, in which the the RNA and these phenomena are not defined as catalytic activity of a key enzyme in a metabolic pathway is allostery. Given the significant structural and functional induced or inhibited upon binding of an allosteric effector potential for catalytic RNAs [S-7], there would also appear molecule. Allosteric regulation of protein enzymes involves to be significant potential for nucleic acids to function as allosteric binding sites, located apart from the active site of allosteric enzymes. For example, RNA and DNA aptamers the enzyme, that can specifically recognize effector mol- have the capacity to form distinct secondary and tertiary ecules. Binding of the effector molecule to the allosteric structures that can function as receptors, binding a variety binding site induces conformational changes in the protein of small organic ligands with considerable affinity and structure that alter the catalytic rate of the enzyme. specificity [17-191. These aptamers can be manipulated as modular receptor elements, a characteristic that has been Natural ribozymes [l-4] and ribozymes that have been exploited for the in vitro selection of novel ribozymes that isolated by in vitro selection [S-7], are not known to make use of pre-existing ligand-binding domains [20,21]. operate as true allosteric enzymes. The activity of natural catalytic RNAs, however, can be greatly affected by spe- Certain characteristics of ribozyme function can be ‘engi- cific proteins that support proper structural folding [8-131. neered’ in a rational fashion by judicious use of simple 454 Chemistry & Biology 1997, Vol 4 No 6 Figure 1 Table 1 Catalytic rates of various ribozyme constructs. (a) AGAG kobs (min-1) HZ C-Go-c- -. Construct Stem II None ATP dATP 32”=; l Gp G-C HI 0.58 - H2 0.10 - CAAC H3* III1 0.054 0.0003 0.053 (b) H4 GUUG c AGAG CAAC H4+ I I I I 0.042 0.061 GUUG CAAGGCC H5* lllllll 0.075 0.13 GUUCCGG CGUAUGC H6* I**I**I 0.022 0.12 0.027 GUGUGUG CGUGUGC CCG H7* I . I 0.0012 0.0098 0.0009 H5 GUGUGUG Hammerhead ribozyme constructs. (a) Hl (shown bracketed) is *Constructs containing a functional ATP aptamer. +Constructs ‘HH15’, originally characterized by Fedor and Uhlenbeck [40]. Roman containing a defective ATP aptamer. Constructs H6 and H7 are numerals identify stems that form the hammerhead secondary identical to H5 except for the indicated changes in stem II. -, Kinetic structure. Stem II is also indicated in red. Construct H2 carries an values not determined. additional G-C base pair in stem I and is flanked on each end by accessory sequences that are designed as short hairpins to reduce the occurrence of inactive structures. (b) Construct H3 is an integrated ribonucleoside triphosphates. This aptamer also under- hammerhead ribozyme that includes an ATP-specific and adenosine- goes a significant conformational change upon ligand specific aptamer (shown in blue and red) [24]. Constructs H4 and H5 are modified versions of H3 that include an aptamer-domain mutation binding, as determined by chemical probing studies. We and a three base-pair extension of stem II, respectively. Arrowheads reasoned that these characteristics might be exploited to indicate the site of ribozyme-mediated cleavage. create a conjoined aptamer-ribozyme molecule that could be subject to ATP-dependent allosteric control. design strategies that make use of Watson-Crick base- Results and discussion pairing rules, thermodynamic stability of RNA duplexes, We used the bimolecular hammerhead ribozyme construct conformational changes, and the modular use of RNA Hl as a basis for the design of new constructs (Figure 1). functional domains [5,6,22]. Here, using these simple First, we appended sequences at the 5’ and 3’ termini to rational design concepts, we joined aptamer domains with make the new constructs amenable to amplification by hammerhead self-cleaving ribozymes [23], to create a reverse transcription-polymerase chain reaction methods series of catalytic RNAs that are amenable either to nega- for use in subsequent studies, giving construct HZ tive or to positive allosteric control by small-molecule (Figure la) [25]. Construct H2 also differs from Hl in that effecters. For our initial work, we used the 40-nucleotide it has an additional G-C base pair in stem I. These changes ATP-binding aptamer (ATP40-1) that was described by cause a sixfold reduction in kobs for H2 compared to that of Sassanfar and Szostak [24]. The ATP40-l aptamer shows Hl (Table 1). We next modified stem II to carry the ATP specific affinity for adenosine 5’ triphosphate (ATP; aptamer, giving construct H3 (Figure lb). Our decision to K,-IOpM) and adenosine, but has no detected affinity for locate the aptamer here was made primarily because a variety of ATP analogs including 2’-deoxyadenosine 5’ changes in stem II can have large effects on the catalytic triphosphate (dATP) or the remaining three natural rates of hammerhead ribozymes [26,27]. In the absence of Research Paper Design of allosteric ribozymes Tang and Breaker 455 Figure 2 ATP-mediated and adenosine-mediated inhibition of a hammerhead ribozyme. (a) Hl H2 H3 (a) Hammerhead constructs Hl, H2 and H3 S (400 nM) were incubated with trace amounts + - + + of [5’-szP]-labeled substrate (S) in the absence (-) or presence (+) of 1 mM ATP for 30 min. (b) The specificity of the effector molecule was examined by incubating H3 and S for 45 min as described above in the absence (-) or presence of 1 mM of various nucleotides as indicated. Similarly, constructs H4 and H5 were examined for activity in the presence of 1 mM ATP. Reaction products were separated by PAGE (200/o, denaturing) and visualized by autoradiography. E, S and P identify enzyme, substrate and product bands, respectively. W H3 H4 H5 S ATP, this alteration in H3 causes an additional twofold identical to H3, but carries a G + C mutation that disrupts reduction in rate compared to that observed with Hl. the consensus sequence of the ATP aptamer. This same guanine residue was strictly conserved among the selected The RNA-cleavage activity of H3 is significantly reduced ATP-binding RNAs, and a G-+A mutation at this posi- when incubated with 1 mM ATP (Figure Za). In contrast, tion has already been shown to eliminate ATP binding by ATP has no effect on the cleavage activity of Hl or HZ. the ATP-40-1 aptamer domain [24]. As expected, this Moreover, inhibition is observed in the presence of adeno- mutation eliminates the allosteric inhibition effect of ATP sine, but not with dATP or the other ribonucleoside with H4 (Figure Zb). triphosphates (Figure Zb). This inhibition is highly spe- cific and is consistent with the previously determined We speculated that the allosteric effect observed with H3 binding specificity of the aptamer [24].