- Email: [email protected]
Allosteric nucleic acid catalysts
318
Allosteric nucleic acid catalysts Garrett A Soukup and Ronald R Breaker* Endowing nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA as controllable therapeutic agents or as sensors of their cognate effector compounds. The ability to engineer RNA catalysts that are allosterically regulated by effector binding has been propelled by the union of modular rational design principles and powerful combinatorial strategies. Addresses Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA *e-mail: [email protected] Current Opinion in Structural Biology 2000, 10:318–325 0959-440X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Introduction Nucleic acid catalysts can accelerate a multitude of chemical transformations, the most common involving phosphoester transfer reactions [1•–4•]. Although the active sites of RNA and DNA catalysts usually consist of noncanonical base pairing and tertiary interactions, they are typically organized by peripheral elements of secondary structure. It is widely accepted that the formation of secondary structure nucleates the folding of functional nucleic acid molecules [5,6,7 • ]. Consequently, the alteration of these peripheral elements can affect the folding and function of nucleic acid catalysts. In recent years, structural dynamics in nucleic acid folding has been resolutely employed to engineer allosteric RNA catalysts [8,9]. An allosteric ribozyme, like its protein enzyme counterpart, possesses an effector-binding site that is distinct from the active site of the catalyst. Effector binding modulates the activities of such catalysts by inducing conformational changes in the nucleic acid structure that are either conducive or inhibitory to ribozyme function. Effector-induced conformational changes are typically mediated by a shared structural element that is essential to the effector-binding and catalytic sites of the allosteric ribozyme. Allosteric activation or inhibition of ribozyme activity can be achieved by ligand-mediated folding of the catalyst into an active or inactive conformation. The ability to endow nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA catalysts as controllable therapeutic agents or as sensors of their cognate effector compounds. In the sections that follow, we examine strategies for engineering effectordependent catalysts, explore the characteristic properties and complexities of allosteric ribozymes, and consider the prospects for allosteric nucleic acid catalysts.
Engineering allosteric ribozymes Initial efforts to engineer allosteric ribozymes relied solely on modular rational design strategies. By considering the structural and functional components requisite for an allosteric catalyst, allosteric ribozymes were logically constructed by combining pre-existing ligand-binding and catalytic RNA motifs. For example, the hammerhead ribozyme, a small catalytic motif that cleaves RNA by a transesterification reaction [10,11], has been transformed into an allosteric ribozyme by appending an aptamer domain that specifically binds ATP (Figure 1a) [12–14]. The resulting construct (H3; Table 1) exhibits allosteric inhibition in response to ATP binding. The modular rational design of such an allosteric ribozyme relies heavily upon structural specifications derived from biochemical or biophysical studies of the component functional domains. For example, the ATPbinding aptamer envelops the ligand as an integral component of the aptamer–ligand structure [15]. As aptamer RNA folding is concomitant with ligand binding, such aptamers are said to exhibit adaptive binding [16,17•]. For ATP-inhibited ribozyme H3, proper folding of both the aptamer and the ribozyme domains is mutually exclusive. The mechanism for ATP-dependent inhibition of H3 activity has been modeled to involve a steric clash between the folded aptamer and ribozyme domains (Figure 1b) [18•]. Modular rational design has also been employed to generate allosteric hammerhead ribozymes that are activated by effector binding (Figure 1c) [13,19•,20•]. In this case, proper folding of the aptamer and ribozyme domains is interdependent. Again, the successful design of these allosteric ribozymes requires an understanding of the structural specifications of each functional component. For example, G–C or G•U pairing in stem II, adjacent to the catalytic core of the hammerhead ribozyme, is required for optimal cleavage activity [21,22]. Similarly, ligand binding to the ATP-binding aptamer enforces G–C pairing in the adjacent stem structure [12]. By appending aptamer and ribozyme domains through common stem elements, the proper folding of both domains can be engineered to be dependent upon ligand binding. In this manner, allosteric hammerhead ribozymes that are activated by ATP (construct H7) [13], FMN (constructs Rz3 and H10) [19•,20•,23,24] or theophylline (construct H14) [20•,25,26] have been generated exclusively by modular ration design (Figure 1c; Table 1). The folding and function of nucleic acid catalysts will naturally be susceptible to antisense interactions with complementary oligonucleotides. Indeed, an allosteric ribozyme has been created that is activated by an
Allosteric nucleic acid catalysts Soukup and Breaker
oligonucleotide effector that binds and sequesters an accessory domain of the hammerhead RNA [27]. This accessory domain otherwise promotes misfolding and disruption of the catalytic core. Other allosteric ribozymes that are activated by interaction with RNA or DNA oligonucleotides have been developed using modular rational design principles (Figure 1d) [28••] or have been identified from an in vitro selection experiment designed to evolve ‘RNA ligase’ ribozymes (Figure 1e) [29••]. Interestingly, the L1 RNA ligase isolated by selection has been engineered to respond to both an oligonucleotide and ATP as effector molecules (Figure 1e), demonstrating that similar design principles can be applicable to a wide range of nucleic acid catalysts. Modular rational design will probably continue to be an essential component of efforts to engineer allosteric nucleic acid catalysts; however, combinatorial selection strategies can augment any design effort by providing a diversity of possible solutions, rather than individually testing designed elements [30]. For example, the precise sequence of a stem element that is shared between the aptamer and ribozyme domains affects both folding and allosteric regulation [13,19•,20•]. The replacement of the shared stem element with a random-sequence region generates a population of potential allosteric catalysts and eliminates the necessity for the rational design of a ligandresponsive stem (Figure 2a). Members of the population that contain specific sequence elements that enable allosteric regulation of ribozyme activity in response to effector binding can be identified using iterative in vitro selection techniques [31,32]. The intersection of modular rational design and combinatorial strategies, as described above, has enabled the selection of FMN-sensitive hammerhead ribozymes that are either activated or inhibited by effector binding (Figure 2b) [33••]. These allosteric catalysts outperform FMN-dependent ribozymes generated using modular rational design strategies alone [19•,20•] with respect to the magnitude of the allosteric response (Table 1). Furthermore, the unique stem elements of FMN-activated ribozymes have been demonstrated to function as ‘communication modules’, which convey the binding status of an appended aptamer domain to the adjoining ribozyme domain. One such communication module (cm+FMN1) mediates ligand-dependent activation of the hammerhead ribozyme when the FMN-binding domain is replaced with either an aptamer domain that binds ATP or an aptamer domain that binds theophylline (Figure 2b; Table 1). Therefore, the selection of communication modules (Figure 2a) provides unique sequence solutions for allosteric regulation that might not be intuitively accessible through rational design. Moreover, selection provides novel components for the future modular rational design of allosteric ribozymes. For example, ribozymes with new effector specificities can, in some cases, be created simply by aptamer domain swapping (Figure 2a).
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Modular rational design and combinatorial strategies can also be exploited to generate allosteric ribozymes that respond to ligands for which no aptamer domain has previously been identified. The development of such allosteric ribozymes is achieved by the selection of effector-dependent catalysts from populations of ribozymes in which the aptamer domain has been mutagenized or completely randomized (Figure 2a). For example, the effector specificity of a theophylline-dependent hammerhead ribozyme (Figure 2b) has been altered through mutagenesis of the aptamer domain and subsequent selection for catalysts that are activated by theophylline-related compounds [34••]. Using this process, a related allosteric ribozyme that favors 3-methylxanthine as an effector was identified (Figure 2b). The novel effector specificity of the 3-methylxanthine-dependent ribozyme is attributed to a single point mutation in the aptamer domain that alters molecular recognition. In a similar fashion, new effector-binding domains can be identified from random sequences through selection for allosteric ribozyme function (Figure 2a). From a single population of potential catalysts, allosteric ribozymes that contain novel effector-binding sites for three different cyclic nucleotide monophosphate compounds (cNMPs) have been identified (Figure 2b; Table 1) [35••]. Each cNMPdependent ribozyme discriminately binds its cognate effector. Novel effector-dependent ribozymes and ligandbinding motifs can therefore be identified using powerful combinatorial selection techniques. Furthermore, allosteric selection is unique in that multiple effector specificities can be developed in a single selection effort [34••,35••].
Structural dynamism in nucleic acid allostery Dynamic structural interplay is an obligatory feature of nucleic acid catalysts that exhibit allosteric transitions in response to effector binding. However, this feature can positively or negatively impact certain aspects of allosteric regulation and effector recognition. For example, some allosteric ribozymes are susceptible to misfolding problems, whereby alternative structural conformations that either preclude or are unaffected by ligand binding contribute to ribozymes that require global denaturation and refolding to exhibit an allosteric response [13,19•,20•,29••]. On the other hand, a number of allosteric ribozymes that bind small effector compounds exhibit the capability to rapidly convert from an ‘inactive’ to an ‘active’ state or vice versa upon addition of ligand [33••–35••]. These catalysts demonstrate that effector binding can thermodynamically tip the balance in favor of different folding solutions that define the ‘inactive’ or ‘active’ states. For these allosteric ribozymes, structural dynamics affords a sensitive and immediate means by which effector binding can modulate ribozyme activity. As allosteric transitions in nucleic acid catalysts are dependent upon the conformational changes that accompany effector binding, the unoccupied ligand-binding domain must be only transiently compatible with ligand recognition.
320
Nucleic acids
(a)
G A G C U 3'C
IV
(b)
A
G UC C U G G Hammerhead C G A 5' A U ribozyme p U G IV C G G C H3 G C III G C G C C G GU A I AA UA G U A C C A AC G G G U U G C C C 3' A A G U UGA CCA GC GGGCCG AA GU GUC G A II G A GAA 5'ppp G G C G A
ATP
II I III
ATP aptamer
GA A G UC G C U G C G C G U A 5' A U 3' C U Gp C G H7 IV G G C C III G C G C C G I GU A II A UA G U AA C C G U GU G C G G G U U G C C C 3' A A G U G U G U GA C C AG C G G G C C G A A G U G UC A G A G A AG 5' ppp G G C G A
(c)
ATP aptamer
3' 5'
H14
Theophylline aptamer
FMN aptamer
ABL exon 2
5'
3'
ABL exon 2
BCR exon 2
(e)
C C G A A G A A G G
G G G MzR G C A G U U III U C C C U C U A U II A C A U C GA
C U U C C C
G G A GA A C U CA C GU GUC A I A A G G MzL G
5' 3'
C C U C A G G G UC
U G A G U G 3' 5'
FMN aptamer 3' 5' U Gp G C III G C C G AA UA C U A U GGC GU G A A G G CC G G U U G C C C 3' A A CC G G C C G CC C A G C G G G ppp 5' AUA AG A UA G U I
C G Rz3 C G III U A C G II A UC GA U AU G C G A A U A G C AG A G G A C 3' U U UC G A C G U U G G AG AG C C C U G 5' A U AG U I
(d)
3' 5' U Gp G C III G C C G A AA UA G G C AG A G G U G A G G U U G C C C 3' C U CGU U CC C A G C G G G ppp 5' A U G AG A G U U AG I AG
H10
Oligonucleotide effector
Oligonucleotide effector
5' G C G A C T G G A C A T C A C G A G 3'
5' G C G A C T G G A C A T C A C G A G 3'
3' C G C U G A C C U G U A G U G C U C U 5' C G Substrate- G C U A binding site G C G U 3' A G ppp 5' U G U A G C G U A U G C G A C A G G C G GA A C C UUC A C A UCU U A G A C U G G UG U A C C A G C U UG CG C C UGG UA U C G C G C G G U G C G C G C A C U A U A C G U C C G A U A G L1 ligase C G AC G G C U U GC
3' C G C U G A C C U G U A G U G C U C U 5' C G Substrate- G C U A binding site G C G U 3' A G ppp 5' UC U G U G U A C G G C A U G U C G A U G C G C G C G GU A A G U C C G GU CU UA G A C A A G U U A C C A G C U UG G A C G G A AG C G G C ATP aptamer A G AA
L1.dB2-ATPm1
Current Opinion in Structural Biology
The inherent lack of pre-organization of the ligand-binding site is expected to diminish the effector-binding affinity of the allosteric ribozyme. Indeed, allosteric ribozymes engi-
neered to respond to ATP [13], FMN [19•,20•,33••] and theophylline [34••] exhibit apparent dissociation constants (apparent Kd) for their cognate ligands that are larger than
Allosteric nucleic acid catalysts Soukup and Breaker
321
Figure 1 Modular rational design of allosteric ribozymes. (a) An ATP-inhibited allosteric hammerhead ribozyme [13]. Nucleotides that comprise the aptamer domain (gray) and hammerhead ribozyme domain (black) are shown. Stem structures that organize the cores of the ribozyme and aptamer domains are numbered I–IV and the site of cleavage is indicated by an arrowhead. (b) Structural model of an ATP-inhibited ribozyme [18•]. A ribbon diagram traces the phosphodiester backbone of the hammerhead domain (black) and ATP-binding aptamer domain (gray). A steric clash between stem I of the hammerhead domain and stem IV of the aptamer domain is evident in the ATP-bound structure. The approximate site of ATP binding is indicated. (c) Ligand-activated allosteric hammerhead ribozymes. Allosteric ribozymes that are
activated by ATP (H7) [13], FMN (H10 and Rz3) [19•,20•] or theophylline (H14) [20•] binding are depicted. In each case, the aptamer domain is appended to stem II of the hammerhead ribozyme. (d) An oligonucleotide-dependent hammerhead ribozyme [28••]. A ribozyme domain composed of two RNAs (black) binds to the mRNA product (gray) of a BCR–ABL gene fusion. The mRNA serves as both allosteric effector and substrate for the allosteric ribozyme. (e) Allosteric ligase ribozymes [29••]. Oligonucleotide effector binding to the L1 ligase is required for activity. An arrowhead indicates the site of ligation between the substrate (gray) and ribozyme (black). The L1.dB2-ATPm1 ribozyme requires both oligonucleotide and ATP for activated ligation.
the Kd values of the respective aptamers. This relationship between ligand-binding affinity and the conformational preorganization of ligand and receptor to form complementary binding surfaces has previously been observed in certain protein–ligand interactions [36–38] and RNA aptamer–ligand interactions [39•]. Although structural dynamism is a necessary component of ribozyme allostery, its adverse affects on folding and ligand-binding affinity can be minimized in order to engineer optimal performance in allosteric nucleic acid catalysis [20•,33••,34••].
‘selfish’ molecules that utilize aberrant folding strategies to survive the selection criteria. Selfish molecules can severely reduce the efficiency of allosteric selection and hamper the isolation of rare ribozyme variants. With additional effort, molecules that exhibit selfish behavior can be disfavored using more stringent or alternative selection strategies [35••]. Future endeavors to create effector-dependent catalysts using allosteric selection techniques must more effectively eliminate selfish RNA species.
The utility of allosteric nucleic acid catalysts will ultimately rely on the ease of engineering specific allosteric enzymes for specified tasks. The ability to generate catalysts that recognize virtually any effector molecule or signal will undoubtedly benefit from allosteric selection techniques [34••,35••]. Efforts to create novel effector-dependent ribozymes using allosteric selection can be complicated by
The dynamic character of allosteric ribozymes will certainly contribute to the utility of such catalysts as biosensors of their cognate ligands. The dynamic range [29••,35••] of an allosteric ribozyme is defined by the magnitude of the allosteric response and the effector concentration range that elicits that response. The magnitude of an allosteric response will probably be limited by the kinetic properties of the catalytic motif. Specifically, an allosteric response may
Table 1 Allosteric ribozymes. Allosteric effect
Activation
Inhibition
Effector specificity
Ribozyme motif
Fold modulation*
Construct name
References
Oligonucleotide ATP FMN FMN Theophylline Oligonucleotide Oligonucleotide Oligonucleotide and ATP FMN Theophylline ATP cGMP cCMP cAMP Theophylline FMN 3-Methylxanthine ATP Theophylline FMN
Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead L1 ligase L1 ligase Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead
10 8 6 100 40 – 10,000 800 270 110 40 5000 500 300 2300 – – 180 5 250
– H7 Rz3 H10 H14 MzL/R L1 L1.dB2-ATPm1 cm+FMN1† cm+FMN1† cm+FMN1† cGMP-1 cCMP-1 cAMP-1 cm+theo5† cm+theo5† cm+theo5† H3 H8 cm–FMN2†
[27] [13] [19•] [20•] [20•] [28••] [29••] [29••] [33••] [33••] [33••] [35••] [35••] [35••] [34••] [34••] [34••] [13,18•] [13] [33••]
*Fold modulation reflects the ratio of catalytic rate constants determined in the presence (k+) and absence (k–) of effector. For allosteric activation and inhibition, fold modulation is k+/k– and k–/k+, respectively. Dashes indicate values not determined. †Constructs consist of the corresponding aptamer values and ribozyme domains interacting through the communication module designated (see Figure 2b).
322
Nucleic acids
(a)
Communication module selection
i
Aptamer domain swapping
ii
Mutagenized aptamer (allosteric selection)
iii
Random sequence (allosteric selection)
(b)
i
cm+FMN1
AA G A U G G C III G C C G A A UA GA
cm+FMN1
C C UU G G C A G A C G U C A A GGC A CCG CC A U U C C GA G G A AU
II
U AG U
C
Nn
GA G
cm–FMN2
UC U G AA C G G A A U U G C G G C cm+FMN1 G C III G C G C C G GU A A UA U G AA C C A C GUC G G G U U G C C C 3' A A G U U C C GA C C A G C G G G ppp 5' G U GU C G A I A II G A AG
G G U U G C C C 3'
C C C A G C G G G ppp 5'
I
ATP aptamer
AA G A U G G C III G C C G AA UA GA
II
I
FMN aptamer
cm+theo5
C C UU G G C A G U U A G A C A A G GC A CC G CC A G G U C C GA G A AU G
C C A G C G G G ppp 5'
G A A GG C UC U C G G G U U G C C C 3' C GC A U C GU A G G U A AG U C C C A G C G G G ppp 5' A U GG U AG U A I II
ii
cm+theo5
G G U U G C C C 3'
ii
Theophylline aptamer
i
C U AG U
Ribozyme domain
AA G A U G G C III G C C G A A UA GA
i
FMN aptamer
AA G A U G G C III G C C G A A UA GA
Nn
II
iv
G A AGGAC GUC G G G U U G C C C 3' C GCA U C GU A G U U C C AG U C C C A G C G G G ppp 5' AU GG U AG U A I II
ii
Ligandbinding domain
AA G A U G G C III G C C G AA U A GA
U AG
AA G A U G G C III G C C G AA UA GA
G A A GG U UAG AC G G G U U G C C C 3' C GC A U C GU A G G C C A G C G G G ppp 5' U C C G A U G C AU G GU GU A A I
FMN aptamer
II
G G U U G C C C 3'
C C A G C G G G ppp 5' UC
I
AA G A U G G C III G C C G A A UA GA
iii
cm+theo5
Theophylline aptamer
C A UU G GC A G U U A G A C A A G GC A CCG A G G U C C GA CC G AU A G
II
3-Methylxanthine aptamer iv
AA G A U G AA G A G A G C U G U A G C G C III G C cCMP-1 U A C G G C A A A UA U AA C G G U U G C C C 3' CG C G C C A G C G G G ppp 5' GA C G C G U AG U I GA C U UU
iv AA GA C G A U G U G C A G C G C III G C cGMP-1 U A C G G CA AA U A G UC GA G G U U G C C C 3' A A C C A G C G G G ppp 5' G A C A C GU GU A I A UC A U C G G C U G A
cCMP aptamer
U AG U
G G U U G C C C 3'
CC C A G C G G G ppp 5'
I
CAU A G AA C A G A G C U G G U G C U A III G C cAMP-1 G C C G C G A G A UA U A AA G G U U G C C C 3' CG C A GA C C A G C G G G ppp 5' A C C G U AG U I A C GG U GU
iv
cAMP aptamer
cGMP aptamer Current Opinion in Structural Biology
Allosteric nucleic acid catalysts Soukup and Breaker
323
Figure 2 Modular rational design and combinatorial selection strategies for generating allosteric ribozymes. (a) Four different strategies include (i) communication module selection [33••,34••], (ii) aptamer domain swapping [33••], (iii) altering the effector specificity of existing aptamer domains using allosteric selection [34••] and (iv) identifying novel effector-binding domains from random sequences using allosteric selection [35••] (see text for details). (b) Allosteric ribozymes engineered by communication module selection, aptamer domain swapping or allosteric selection. The strategy used to develop each
construct is indicated. Communication modules (shaded) are named according to whether they convey allosteric activation (cm+) or inhibition (cm–), and the effector specificity with which they were originally developed to function [35••]. Arrows indicate related constructs developed by aptamer domain swapping or allosteric selection for altered effector specificity. A single point mutation in the 3-methylxanthine aptamer that accounts for its altered specificity relative to the theophylline aptamer is indicated (shaded).
not exceed the ratio of the maximum rate constant of the catalyst to the rate of the uncatalyzed reaction. For example, although the rate constant for hammerhead-ribozyme-catalyzed transesterification is approximately 1 min–1 under near physiological conditions [21], the rate constant for uncatalyzed RNA transesterification under similar conditions is approximately 10–7 min–1 [40]. Therefore, the maximal allosteric response that could be achieved for this ribozyme is seven orders of magnitude. Several allosteric hammerhead ribozymes exhibit dynamic ranges of three to four orders of magnitude (Table 1) [29••,34••,35••]. Allosteric ribozymes with extended dynamic ranges would offer even greater versatility and sensitivity in the detection of their cognate effector compounds.
of a reporter gene construct in vivo, as well as alleviate the effect of an oncogene product in normal and leukemic cells. Allosteric ribozymes therefore possess potential as controllable therapeutic agents for gene therapy strategies. Furthermore, allosteric catalysts could be used as molecular biological tools for temporally manipulating the expression of gene products in cells in order to examine function.
Prospects for allosteric nucleic acid catalysts As described above, allosteric ribozymes are innate sensors of their cognate effector compounds because they catalyze specific reactions in response to effector binding. The general utility of allosteric catalysts as biosensors will largely depend on the capability of nucleic acids to recognize intended target molecules with great specificity and affinity. Of additional importance to their general utility is the ease with which such catalysts can be developed. Already, RNA has demonstrated an immense capacity to bind a variety of target compounds with high affinity and specificity [41,42,43•] and allosteric selection strategies have indicated that novel effector specificities for allosteric ribozymes can be developed in a massively parallel fashion [34••,35••]. Furthermore, allosteric catalysts can be employed to report the presence or concentration of specific effectors in any one of a number of ways. Although catalysis is a direct measure of the effector modulation of allosteric ribozymes, schemes in which the catalytic event is coupled to amplification, accessory reporters or microchip technologies have been demonstrated [29••] or envisioned [44•]. Allosteric ribozymes could also be used as molecular switches for the control of RNA function in cells. For example, allosteric ribozymes can be utilized to control gene expression by targeting the destruction of mRNAs in response to effector binding. Indeed, an oligonucleotidedependent hammerhead ribozyme (construct MzL/R; Figure 1d) has been developed for which a specific mRNA serves as both effector and substrate [28••]. This allosteric ribozyme has been demonstrated to inhibit the expression
Allosteric ribozymes also provide an opportunity to examine molecular recognition and structural dynamism in nucleic acid structures. Already, the mechanistic function of certain communication modules has been proposed to involve specific local base-pairing rearrangements in response to effector binding [33••]. Furthermore, the evolution of ligand-binding specificities in aptamers that bind theophylline or 3-methylxanthine has revealed the intricacies of molecular recognition in these related aptamer motifs [34••]. The further development of novel allosteric ribozymes will undoubtedly offer additional opportunities to examine molecular recognition and structural dynamism in nucleic acid structure.
Conclusions The development of allosteric nucleic acid catalysts has been accelerated by the intersection of modular rational design principles and combinatorial selection strategies. Engineering allosteric ribozymes that respond to specific effector stimuli will probably become a routine enterprise for creating potential therapeutic agents or molecular switches that are controlled by specific chemical or physical signals. These molecular switches could find specific application in the artificial control of gene expression in cells. Additionally, allosteric ribozymes may find utility as sensors for the detection of virtually any target compound or signal. These prospects for allosteric nucleic acid catalysts will be met with anticipation, as future endeavors apply the recent advances that have been made in allosteric ribozyme engineering.
Update The L1 ligase ribozyme [29••] has recently been engineered to respond to other molecular effectors, such as FMN and theophylline [45]. This study further demonstrates that modular rational design, communication module selection and aptamer domain swapping can be generally applied to the development of allosteric nucleic acid catalysts.
324
Nucleic acids
Acknowledgements We thank GAM Emilsson for critical reading of the manuscript and other members of the Breaker laboratory for helpful discussions. This work was supported by research grants from the National Institutes of Health (GM559343), DARPA and the Yale Diabetes Endocrine Research Center (DERC). Support is also provided by fellowships to RRB from the Hellman Family and from the David and Lucile Packard Foundation.
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the aptamer and ribozyme domains. This study demonstrates how biophysical and biochemical data can together yield a clear mechanistic understanding of allosteric ribozyme function. 19. Araki M, Okuno Y, Hara Y, Sugiura Y: Allosteric regulation of a • ribozyme activity through ligand induced conformational change. Nucleic Acids Res 1998, 26:3379-3384. See annotation to [20•]. 20. Soukup GA, Breaker RR: Design of allosteric hammerhead • ribozymes activated by ligand-induced structure stabilization. Structure 1999, 7:783-791. The work described here and in [19•] similarly employ modular rational design techniques [13] to generate allosteric ribozymes that are responsive to FMN. 21. Fedor MJ, Uhlenbeck OC: Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry 1992, 31:12042-12054. 22. Ruffner DE, Stormo GD, Uhlenbeck OC: Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 1990, 29:10695-10702. 23. Burgstaller P, Famulok M: Isolation of RNA aptamers for biological cofactors by in vitro selection. Angew Chem Int Ed Engl 1995, 33:1084-1087. 24. Fan P, Suri AK, Fiala R, Live D, Patel DJ: Molecular recognition in the FMN–RNA aptamer complex. J Mol Biol 1996, 258:480-500. 25. Jenison RD, Gill SC, Pardi A, Polisky B: High resolution molecular discrimination by RNA. Science 1994, 263:1425-1429. 26. Zimmermann GR, Jenison RD, Wick CL, Simorre JP, Pardi A: Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA. Nat Struct Biol 1997, 4:644-649. 27.
Porta H, Lizardi PM: An allosteric hammerhead ribozyme. Biotechnology 1995, 13:161-164.
28. Kuwabara T, Warashina M, Tanabe T, Tani K, Asano S, Taira K: A novel •• allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo. Mol Cell 1998, 2:617-627. This study demonstrates the utility of allosteric ribozymes as controllable therapeutic agents for modulating gene expression in vivo. Although the effector and substrate mRNA for this oligonucleotide-dependent allosteric hammerhead ribozyme construct is endogenous to the target cells, allosteric ribozymes that respond to exogenous effector molecules (i.e. pharmaceutical agents) may function similarly to control mRNA stability. 29. Robertson MP, Ellington AD: In vitro selection of an allosteric •• ribozyme that transduces analytes to amplicons. Nat Biotechnol 1999, 17:62-66. An oligonucleotide-dependent allosteric RNA ligase ribozyme identified from an in vitro selection experiment is used to demonstrate how allosteric catalysts can be used to detect low concentrations of effector molecules. Effector concentration can be determined directly from the catalytic activity of the ribozyme or by selective amplification of the resulting ribozyme ligation product.
11. Haseloff JP, Gerlach WL: Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 1988, 334:585-591.
30. Breaker RR, Joyce GF: Inventing and improving ribozyme function: rational design versus iterative selection methods. Trends Biotechnol 1994, 12:268-275.
12. Sassanfar M, Szostak JW: An RNA motif that binds ATP. Nature 1993, 364:550-553.
31. Williams KP, Bartel DP: In vitro selection of catalytic RNA. Nucleic Acids Mol Biol 1996, 10:367-381.
13. Tang J, Breaker RR: Rational design of allosteric ribozymes. Chem Biol 1997, 4:453-459.
32. Osborne SE, Ellington AD: Nucleic acid selection and the challenge of combinatorial chemistry. Chem Rev 1997, 97:349-370.
14. Tang J, Breaker RR: Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection. RNA 1997, 3:914-925.
33. Soukup GA, Breaker RR: Engineering precision RNA molecular •• switches. Proc Natl Acad Sci USA 1999, 96:3584-3589. The intersection of modular rational design and combinatorial selection techniques was used to isolate novel allosteric catalysts that contain unique sequence elements or ‘communication modules’ that function between an aptamer domain and the hammerhead ribozyme. Furthermore, certain communication modules exhibit the ability to control ribozyme activity in response to effector binding, regardless of the sequence or ligand specificity of the appended aptamer domain.
15. Jiang F, Kumar RA, Jones RA, Patel DJ: Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex. Nature 1996, 382:183-186. 16. Patel DJ, Suri AK, Jiang F, Jiang L, Fan P, Kumar RA, Nonin S: Structure, recognition and adaptive binding in RNA aptamer complexes. J Mol Biol 1997, 272:645-664. 17. Hermann T, Patel DJ: Adaptive recognition by nucleic acid • aptamers. Science 2000, 287:820-825. A comprehensive review of RNA–ligand interactions. The molecular interactions that dictate discriminatory ligand recognition by RNA aptamers are examined in detail. 18. Tang J, Breaker RR: Mechanism for allosteric inhibition of an • ATP-sensitive ribozyme. Nucleic Acids Res 1998, 26:4222-4229. A detailed analysis of the ATP-inhibited hammerhead ribozyme reveals a mechanism of allosteric regulation that involves steric interactions between
34. Soukup GA, Emilsson GAM, Breaker RR: Altering molecular •• recognition of RNA aptamers by allosteric selection. J Mol Biol 2000, 298:623-632. By mutagenizing the aptamer domain of an allosteric ribozyme, the ligand specificity of an existing aptamer can be altered by performing allosteric selection [35••] for catalysts that are activated by compounds related to the cognate ligand. The identification of related aptamer–ligand interactions provides an opportunity to examine the details of molecular recognition and discrimination in RNA–ligand complexes, as well as novel effector-dependent catalysts.
Allosteric nucleic acid catalysts Soukup and Breaker
35. Koizumi M, Soukup GA, Kerr JNQ, Breaker RR: Allosteric selection •• of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Biol 1999, 6:1062-1071. The potential to generate allosteric ribozymes that respond to virtually any effector compound or signal is demonstrated. By attaching a random sequence region to the hammerhead ribozyme, allosteric catalysts that are activated by any one of a number of effector compounds can be developed simultaneously through allosteric selection, whereby novel ligand-binding domains are derived from the random sequence region. 36. Van Duyne GD, Standaert RF, Karplus PA, Schreiber SL, Clardy J: Atomic structure of FKBP-FK506, an immunophilinimmunosuppressant complex. Science 1991, 252:839-852. 37.
Katz BA: Binding to protein targets of peptidic leads discovered by phage display: crystal structures of streptavidin-bound linear and cyclic peptide ligands containing the HPQ sequence. Biochemistry 1995, 34:15421-15429.
38. Giebel LB, Cass R, Milligan DL, Young D, Arze R, Johnson C: Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities. Biochemistry 1995, 34:15430-15435. 39. Sussman D, Nix JC, Wilson C: The structural basis for molecular • recognition by the vitamin B12 RNA aptamer. Nat Struct Biol 2000, 7:53-57. Unlike other RNA aptamers that bind small molecules, the cyanocobalamin (vitamin B12)-binding aptamer does not encapsulate ligand as an integral
325
component of the RNA fold. Rather, the RNA structure provides a complementary binding surface for the ligand. The structural pre-organization of this aptamer RNA probably contributes to its relatively high affinity for cyanocobalamin in comparison to other small molecule–aptamer interactions. 40. Li Y, Breaker RR: Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group. J Am Chem Soc 1999, 121:5364-5372. 41. Gold L, Polinsky B, Uhlenbeck OC, Yarus M: Diversity of oligonucleotide functions. Annu Rev Biochem 1995, 64:763-797. 42. Chow CS, Bogdan FM: A structural basis for RNA-ligand interactions. Chem Rev 1997, 97:1489-1513. 43. Famulok M: Oligonucleotide aptamers that recognize small • molecules. Curr Opin Struct Biol 1999, 9:324-329. A current and comprehensive review of small molecule–aptamer interactions. The aptitude of RNA to achieve high affinity for and specific molecular recognition of a variety of target ligands is apparent. 44. Marshall KA, Ellington AD: Training ribozymes to switch. Nat Struct • Biol 1999, 6:992-994. The prospect of utilizing allosteric ribozymes in microchip arrays to concurrently assess the presence or concentration of difference metabolites or effector compounds is described in brief. 45. Robertson MP, Ellington MD: Design and optimization of effectoractivated ribozyme ligases. Nucleic Acids Res 2000, 28:1751-1759.
Allosteric nucleic acid catalysts Garrett A Soukup and Ronald R Breaker* Endowing nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA as controllable therapeutic agents or as sensors of their cognate effector compounds. The ability to engineer RNA catalysts that are allosterically regulated by effector binding has been propelled by the union of modular rational design principles and powerful combinatorial strategies. Addresses Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA *e-mail: [email protected] Current Opinion in Structural Biology 2000, 10:318–325 0959-440X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Introduction Nucleic acid catalysts can accelerate a multitude of chemical transformations, the most common involving phosphoester transfer reactions [1•–4•]. Although the active sites of RNA and DNA catalysts usually consist of noncanonical base pairing and tertiary interactions, they are typically organized by peripheral elements of secondary structure. It is widely accepted that the formation of secondary structure nucleates the folding of functional nucleic acid molecules [5,6,7 • ]. Consequently, the alteration of these peripheral elements can affect the folding and function of nucleic acid catalysts. In recent years, structural dynamics in nucleic acid folding has been resolutely employed to engineer allosteric RNA catalysts [8,9]. An allosteric ribozyme, like its protein enzyme counterpart, possesses an effector-binding site that is distinct from the active site of the catalyst. Effector binding modulates the activities of such catalysts by inducing conformational changes in the nucleic acid structure that are either conducive or inhibitory to ribozyme function. Effector-induced conformational changes are typically mediated by a shared structural element that is essential to the effector-binding and catalytic sites of the allosteric ribozyme. Allosteric activation or inhibition of ribozyme activity can be achieved by ligand-mediated folding of the catalyst into an active or inactive conformation. The ability to endow nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA catalysts as controllable therapeutic agents or as sensors of their cognate effector compounds. In the sections that follow, we examine strategies for engineering effectordependent catalysts, explore the characteristic properties and complexities of allosteric ribozymes, and consider the prospects for allosteric nucleic acid catalysts.
Engineering allosteric ribozymes Initial efforts to engineer allosteric ribozymes relied solely on modular rational design strategies. By considering the structural and functional components requisite for an allosteric catalyst, allosteric ribozymes were logically constructed by combining pre-existing ligand-binding and catalytic RNA motifs. For example, the hammerhead ribozyme, a small catalytic motif that cleaves RNA by a transesterification reaction [10,11], has been transformed into an allosteric ribozyme by appending an aptamer domain that specifically binds ATP (Figure 1a) [12–14]. The resulting construct (H3; Table 1) exhibits allosteric inhibition in response to ATP binding. The modular rational design of such an allosteric ribozyme relies heavily upon structural specifications derived from biochemical or biophysical studies of the component functional domains. For example, the ATPbinding aptamer envelops the ligand as an integral component of the aptamer–ligand structure [15]. As aptamer RNA folding is concomitant with ligand binding, such aptamers are said to exhibit adaptive binding [16,17•]. For ATP-inhibited ribozyme H3, proper folding of both the aptamer and the ribozyme domains is mutually exclusive. The mechanism for ATP-dependent inhibition of H3 activity has been modeled to involve a steric clash between the folded aptamer and ribozyme domains (Figure 1b) [18•]. Modular rational design has also been employed to generate allosteric hammerhead ribozymes that are activated by effector binding (Figure 1c) [13,19•,20•]. In this case, proper folding of the aptamer and ribozyme domains is interdependent. Again, the successful design of these allosteric ribozymes requires an understanding of the structural specifications of each functional component. For example, G–C or G•U pairing in stem II, adjacent to the catalytic core of the hammerhead ribozyme, is required for optimal cleavage activity [21,22]. Similarly, ligand binding to the ATP-binding aptamer enforces G–C pairing in the adjacent stem structure [12]. By appending aptamer and ribozyme domains through common stem elements, the proper folding of both domains can be engineered to be dependent upon ligand binding. In this manner, allosteric hammerhead ribozymes that are activated by ATP (construct H7) [13], FMN (constructs Rz3 and H10) [19•,20•,23,24] or theophylline (construct H14) [20•,25,26] have been generated exclusively by modular ration design (Figure 1c; Table 1). The folding and function of nucleic acid catalysts will naturally be susceptible to antisense interactions with complementary oligonucleotides. Indeed, an allosteric ribozyme has been created that is activated by an
Allosteric nucleic acid catalysts Soukup and Breaker
oligonucleotide effector that binds and sequesters an accessory domain of the hammerhead RNA [27]. This accessory domain otherwise promotes misfolding and disruption of the catalytic core. Other allosteric ribozymes that are activated by interaction with RNA or DNA oligonucleotides have been developed using modular rational design principles (Figure 1d) [28••] or have been identified from an in vitro selection experiment designed to evolve ‘RNA ligase’ ribozymes (Figure 1e) [29••]. Interestingly, the L1 RNA ligase isolated by selection has been engineered to respond to both an oligonucleotide and ATP as effector molecules (Figure 1e), demonstrating that similar design principles can be applicable to a wide range of nucleic acid catalysts. Modular rational design will probably continue to be an essential component of efforts to engineer allosteric nucleic acid catalysts; however, combinatorial selection strategies can augment any design effort by providing a diversity of possible solutions, rather than individually testing designed elements [30]. For example, the precise sequence of a stem element that is shared between the aptamer and ribozyme domains affects both folding and allosteric regulation [13,19•,20•]. The replacement of the shared stem element with a random-sequence region generates a population of potential allosteric catalysts and eliminates the necessity for the rational design of a ligandresponsive stem (Figure 2a). Members of the population that contain specific sequence elements that enable allosteric regulation of ribozyme activity in response to effector binding can be identified using iterative in vitro selection techniques [31,32]. The intersection of modular rational design and combinatorial strategies, as described above, has enabled the selection of FMN-sensitive hammerhead ribozymes that are either activated or inhibited by effector binding (Figure 2b) [33••]. These allosteric catalysts outperform FMN-dependent ribozymes generated using modular rational design strategies alone [19•,20•] with respect to the magnitude of the allosteric response (Table 1). Furthermore, the unique stem elements of FMN-activated ribozymes have been demonstrated to function as ‘communication modules’, which convey the binding status of an appended aptamer domain to the adjoining ribozyme domain. One such communication module (cm+FMN1) mediates ligand-dependent activation of the hammerhead ribozyme when the FMN-binding domain is replaced with either an aptamer domain that binds ATP or an aptamer domain that binds theophylline (Figure 2b; Table 1). Therefore, the selection of communication modules (Figure 2a) provides unique sequence solutions for allosteric regulation that might not be intuitively accessible through rational design. Moreover, selection provides novel components for the future modular rational design of allosteric ribozymes. For example, ribozymes with new effector specificities can, in some cases, be created simply by aptamer domain swapping (Figure 2a).
319
Modular rational design and combinatorial strategies can also be exploited to generate allosteric ribozymes that respond to ligands for which no aptamer domain has previously been identified. The development of such allosteric ribozymes is achieved by the selection of effector-dependent catalysts from populations of ribozymes in which the aptamer domain has been mutagenized or completely randomized (Figure 2a). For example, the effector specificity of a theophylline-dependent hammerhead ribozyme (Figure 2b) has been altered through mutagenesis of the aptamer domain and subsequent selection for catalysts that are activated by theophylline-related compounds [34••]. Using this process, a related allosteric ribozyme that favors 3-methylxanthine as an effector was identified (Figure 2b). The novel effector specificity of the 3-methylxanthine-dependent ribozyme is attributed to a single point mutation in the aptamer domain that alters molecular recognition. In a similar fashion, new effector-binding domains can be identified from random sequences through selection for allosteric ribozyme function (Figure 2a). From a single population of potential catalysts, allosteric ribozymes that contain novel effector-binding sites for three different cyclic nucleotide monophosphate compounds (cNMPs) have been identified (Figure 2b; Table 1) [35••]. Each cNMPdependent ribozyme discriminately binds its cognate effector. Novel effector-dependent ribozymes and ligandbinding motifs can therefore be identified using powerful combinatorial selection techniques. Furthermore, allosteric selection is unique in that multiple effector specificities can be developed in a single selection effort [34••,35••].
Structural dynamism in nucleic acid allostery Dynamic structural interplay is an obligatory feature of nucleic acid catalysts that exhibit allosteric transitions in response to effector binding. However, this feature can positively or negatively impact certain aspects of allosteric regulation and effector recognition. For example, some allosteric ribozymes are susceptible to misfolding problems, whereby alternative structural conformations that either preclude or are unaffected by ligand binding contribute to ribozymes that require global denaturation and refolding to exhibit an allosteric response [13,19•,20•,29••]. On the other hand, a number of allosteric ribozymes that bind small effector compounds exhibit the capability to rapidly convert from an ‘inactive’ to an ‘active’ state or vice versa upon addition of ligand [33••–35••]. These catalysts demonstrate that effector binding can thermodynamically tip the balance in favor of different folding solutions that define the ‘inactive’ or ‘active’ states. For these allosteric ribozymes, structural dynamics affords a sensitive and immediate means by which effector binding can modulate ribozyme activity. As allosteric transitions in nucleic acid catalysts are dependent upon the conformational changes that accompany effector binding, the unoccupied ligand-binding domain must be only transiently compatible with ligand recognition.
320
Nucleic acids
(a)
G A G C U 3'C
IV
(b)
A
G UC C U G G Hammerhead C G A 5' A U ribozyme p U G IV C G G C H3 G C III G C G C C G GU A I AA UA G U A C C A AC G G G U U G C C C 3' A A G U UGA CCA GC GGGCCG AA GU GUC G A II G A GAA 5'ppp G G C G A
ATP
II I III
ATP aptamer
GA A G UC G C U G C G C G U A 5' A U 3' C U Gp C G H7 IV G G C C III G C G C C G I GU A II A UA G U AA C C G U GU G C G G G U U G C C C 3' A A G U G U G U GA C C AG C G G G C C G A A G U G UC A G A G A AG 5' ppp G G C G A
(c)
ATP aptamer
3' 5'
H14
Theophylline aptamer
FMN aptamer
ABL exon 2
5'
3'
ABL exon 2
BCR exon 2
(e)
C C G A A G A A G G
G G G MzR G C A G U U III U C C C U C U A U II A C A U C GA
C U U C C C
G G A GA A C U CA C GU GUC A I A A G G MzL G
5' 3'
C C U C A G G G UC
U G A G U G 3' 5'
FMN aptamer 3' 5' U Gp G C III G C C G AA UA C U A U GGC GU G A A G G CC G G U U G C C C 3' A A CC G G C C G CC C A G C G G G ppp 5' AUA AG A UA G U I
C G Rz3 C G III U A C G II A UC GA U AU G C G A A U A G C AG A G G A C 3' U U UC G A C G U U G G AG AG C C C U G 5' A U AG U I
(d)
3' 5' U Gp G C III G C C G A AA UA G G C AG A G G U G A G G U U G C C C 3' C U CGU U CC C A G C G G G ppp 5' A U G AG A G U U AG I AG
H10
Oligonucleotide effector
Oligonucleotide effector
5' G C G A C T G G A C A T C A C G A G 3'
5' G C G A C T G G A C A T C A C G A G 3'
3' C G C U G A C C U G U A G U G C U C U 5' C G Substrate- G C U A binding site G C G U 3' A G ppp 5' U G U A G C G U A U G C G A C A G G C G GA A C C UUC A C A UCU U A G A C U G G UG U A C C A G C U UG CG C C UGG UA U C G C G C G G U G C G C G C A C U A U A C G U C C G A U A G L1 ligase C G AC G G C U U GC
3' C G C U G A C C U G U A G U G C U C U 5' C G Substrate- G C U A binding site G C G U 3' A G ppp 5' UC U G U G U A C G G C A U G U C G A U G C G C G C G GU A A G U C C G GU CU UA G A C A A G U U A C C A G C U UG G A C G G A AG C G G C ATP aptamer A G AA
L1.dB2-ATPm1
Current Opinion in Structural Biology
The inherent lack of pre-organization of the ligand-binding site is expected to diminish the effector-binding affinity of the allosteric ribozyme. Indeed, allosteric ribozymes engi-
neered to respond to ATP [13], FMN [19•,20•,33••] and theophylline [34••] exhibit apparent dissociation constants (apparent Kd) for their cognate ligands that are larger than
Allosteric nucleic acid catalysts Soukup and Breaker
321
Figure 1 Modular rational design of allosteric ribozymes. (a) An ATP-inhibited allosteric hammerhead ribozyme [13]. Nucleotides that comprise the aptamer domain (gray) and hammerhead ribozyme domain (black) are shown. Stem structures that organize the cores of the ribozyme and aptamer domains are numbered I–IV and the site of cleavage is indicated by an arrowhead. (b) Structural model of an ATP-inhibited ribozyme [18•]. A ribbon diagram traces the phosphodiester backbone of the hammerhead domain (black) and ATP-binding aptamer domain (gray). A steric clash between stem I of the hammerhead domain and stem IV of the aptamer domain is evident in the ATP-bound structure. The approximate site of ATP binding is indicated. (c) Ligand-activated allosteric hammerhead ribozymes. Allosteric ribozymes that are
activated by ATP (H7) [13], FMN (H10 and Rz3) [19•,20•] or theophylline (H14) [20•] binding are depicted. In each case, the aptamer domain is appended to stem II of the hammerhead ribozyme. (d) An oligonucleotide-dependent hammerhead ribozyme [28••]. A ribozyme domain composed of two RNAs (black) binds to the mRNA product (gray) of a BCR–ABL gene fusion. The mRNA serves as both allosteric effector and substrate for the allosteric ribozyme. (e) Allosteric ligase ribozymes [29••]. Oligonucleotide effector binding to the L1 ligase is required for activity. An arrowhead indicates the site of ligation between the substrate (gray) and ribozyme (black). The L1.dB2-ATPm1 ribozyme requires both oligonucleotide and ATP for activated ligation.
the Kd values of the respective aptamers. This relationship between ligand-binding affinity and the conformational preorganization of ligand and receptor to form complementary binding surfaces has previously been observed in certain protein–ligand interactions [36–38] and RNA aptamer–ligand interactions [39•]. Although structural dynamism is a necessary component of ribozyme allostery, its adverse affects on folding and ligand-binding affinity can be minimized in order to engineer optimal performance in allosteric nucleic acid catalysis [20•,33••,34••].
‘selfish’ molecules that utilize aberrant folding strategies to survive the selection criteria. Selfish molecules can severely reduce the efficiency of allosteric selection and hamper the isolation of rare ribozyme variants. With additional effort, molecules that exhibit selfish behavior can be disfavored using more stringent or alternative selection strategies [35••]. Future endeavors to create effector-dependent catalysts using allosteric selection techniques must more effectively eliminate selfish RNA species.
The utility of allosteric nucleic acid catalysts will ultimately rely on the ease of engineering specific allosteric enzymes for specified tasks. The ability to generate catalysts that recognize virtually any effector molecule or signal will undoubtedly benefit from allosteric selection techniques [34••,35••]. Efforts to create novel effector-dependent ribozymes using allosteric selection can be complicated by
The dynamic character of allosteric ribozymes will certainly contribute to the utility of such catalysts as biosensors of their cognate ligands. The dynamic range [29••,35••] of an allosteric ribozyme is defined by the magnitude of the allosteric response and the effector concentration range that elicits that response. The magnitude of an allosteric response will probably be limited by the kinetic properties of the catalytic motif. Specifically, an allosteric response may
Table 1 Allosteric ribozymes. Allosteric effect
Activation
Inhibition
Effector specificity
Ribozyme motif
Fold modulation*
Construct name
References
Oligonucleotide ATP FMN FMN Theophylline Oligonucleotide Oligonucleotide Oligonucleotide and ATP FMN Theophylline ATP cGMP cCMP cAMP Theophylline FMN 3-Methylxanthine ATP Theophylline FMN
Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead L1 ligase L1 ligase Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead Hammerhead
10 8 6 100 40 – 10,000 800 270 110 40 5000 500 300 2300 – – 180 5 250
– H7 Rz3 H10 H14 MzL/R L1 L1.dB2-ATPm1 cm+FMN1† cm+FMN1† cm+FMN1† cGMP-1 cCMP-1 cAMP-1 cm+theo5† cm+theo5† cm+theo5† H3 H8 cm–FMN2†
[27] [13] [19•] [20•] [20•] [28••] [29••] [29••] [33••] [33••] [33••] [35••] [35••] [35••] [34••] [34••] [34••] [13,18•] [13] [33••]
*Fold modulation reflects the ratio of catalytic rate constants determined in the presence (k+) and absence (k–) of effector. For allosteric activation and inhibition, fold modulation is k+/k– and k–/k+, respectively. Dashes indicate values not determined. †Constructs consist of the corresponding aptamer values and ribozyme domains interacting through the communication module designated (see Figure 2b).
322
Nucleic acids
(a)
Communication module selection
i
Aptamer domain swapping
ii
Mutagenized aptamer (allosteric selection)
iii
Random sequence (allosteric selection)
(b)
i
cm+FMN1
AA G A U G G C III G C C G A A UA GA
cm+FMN1
C C UU G G C A G A C G U C A A GGC A CCG CC A U U C C GA G G A AU
II
U AG U
C
Nn
GA G
cm–FMN2
UC U G AA C G G A A U U G C G G C cm+FMN1 G C III G C G C C G GU A A UA U G AA C C A C GUC G G G U U G C C C 3' A A G U U C C GA C C A G C G G G ppp 5' G U GU C G A I A II G A AG
G G U U G C C C 3'
C C C A G C G G G ppp 5'
I
ATP aptamer
AA G A U G G C III G C C G AA UA GA
II
I
FMN aptamer
cm+theo5
C C UU G G C A G U U A G A C A A G GC A CC G CC A G G U C C GA G A AU G
C C A G C G G G ppp 5'
G A A GG C UC U C G G G U U G C C C 3' C GC A U C GU A G G U A AG U C C C A G C G G G ppp 5' A U GG U AG U A I II
ii
cm+theo5
G G U U G C C C 3'
ii
Theophylline aptamer
i
C U AG U
Ribozyme domain
AA G A U G G C III G C C G A A UA GA
i
FMN aptamer
AA G A U G G C III G C C G A A UA GA
Nn
II
iv
G A AGGAC GUC G G G U U G C C C 3' C GCA U C GU A G U U C C AG U C C C A G C G G G ppp 5' AU GG U AG U A I II
ii
Ligandbinding domain
AA G A U G G C III G C C G AA U A GA
U AG
AA G A U G G C III G C C G AA UA GA
G A A GG U UAG AC G G G U U G C C C 3' C GC A U C GU A G G C C A G C G G G ppp 5' U C C G A U G C AU G GU GU A A I
FMN aptamer
II
G G U U G C C C 3'
C C A G C G G G ppp 5' UC
I
AA G A U G G C III G C C G A A UA GA
iii
cm+theo5
Theophylline aptamer
C A UU G GC A G U U A G A C A A G GC A CCG A G G U C C GA CC G AU A G
II
3-Methylxanthine aptamer iv
AA G A U G AA G A G A G C U G U A G C G C III G C cCMP-1 U A C G G C A A A UA U AA C G G U U G C C C 3' CG C G C C A G C G G G ppp 5' GA C G C G U AG U I GA C U UU
iv AA GA C G A U G U G C A G C G C III G C cGMP-1 U A C G G CA AA U A G UC GA G G U U G C C C 3' A A C C A G C G G G ppp 5' G A C A C GU GU A I A UC A U C G G C U G A
cCMP aptamer
U AG U
G G U U G C C C 3'
CC C A G C G G G ppp 5'
I
CAU A G AA C A G A G C U G G U G C U A III G C cAMP-1 G C C G C G A G A UA U A AA G G U U G C C C 3' CG C A GA C C A G C G G G ppp 5' A C C G U AG U I A C GG U GU
iv
cAMP aptamer
cGMP aptamer Current Opinion in Structural Biology
Allosteric nucleic acid catalysts Soukup and Breaker
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Figure 2 Modular rational design and combinatorial selection strategies for generating allosteric ribozymes. (a) Four different strategies include (i) communication module selection [33••,34••], (ii) aptamer domain swapping [33••], (iii) altering the effector specificity of existing aptamer domains using allosteric selection [34••] and (iv) identifying novel effector-binding domains from random sequences using allosteric selection [35••] (see text for details). (b) Allosteric ribozymes engineered by communication module selection, aptamer domain swapping or allosteric selection. The strategy used to develop each
construct is indicated. Communication modules (shaded) are named according to whether they convey allosteric activation (cm+) or inhibition (cm–), and the effector specificity with which they were originally developed to function [35••]. Arrows indicate related constructs developed by aptamer domain swapping or allosteric selection for altered effector specificity. A single point mutation in the 3-methylxanthine aptamer that accounts for its altered specificity relative to the theophylline aptamer is indicated (shaded).
not exceed the ratio of the maximum rate constant of the catalyst to the rate of the uncatalyzed reaction. For example, although the rate constant for hammerhead-ribozyme-catalyzed transesterification is approximately 1 min–1 under near physiological conditions [21], the rate constant for uncatalyzed RNA transesterification under similar conditions is approximately 10–7 min–1 [40]. Therefore, the maximal allosteric response that could be achieved for this ribozyme is seven orders of magnitude. Several allosteric hammerhead ribozymes exhibit dynamic ranges of three to four orders of magnitude (Table 1) [29••,34••,35••]. Allosteric ribozymes with extended dynamic ranges would offer even greater versatility and sensitivity in the detection of their cognate effector compounds.
of a reporter gene construct in vivo, as well as alleviate the effect of an oncogene product in normal and leukemic cells. Allosteric ribozymes therefore possess potential as controllable therapeutic agents for gene therapy strategies. Furthermore, allosteric catalysts could be used as molecular biological tools for temporally manipulating the expression of gene products in cells in order to examine function.
Prospects for allosteric nucleic acid catalysts As described above, allosteric ribozymes are innate sensors of their cognate effector compounds because they catalyze specific reactions in response to effector binding. The general utility of allosteric catalysts as biosensors will largely depend on the capability of nucleic acids to recognize intended target molecules with great specificity and affinity. Of additional importance to their general utility is the ease with which such catalysts can be developed. Already, RNA has demonstrated an immense capacity to bind a variety of target compounds with high affinity and specificity [41,42,43•] and allosteric selection strategies have indicated that novel effector specificities for allosteric ribozymes can be developed in a massively parallel fashion [34••,35••]. Furthermore, allosteric catalysts can be employed to report the presence or concentration of specific effectors in any one of a number of ways. Although catalysis is a direct measure of the effector modulation of allosteric ribozymes, schemes in which the catalytic event is coupled to amplification, accessory reporters or microchip technologies have been demonstrated [29••] or envisioned [44•]. Allosteric ribozymes could also be used as molecular switches for the control of RNA function in cells. For example, allosteric ribozymes can be utilized to control gene expression by targeting the destruction of mRNAs in response to effector binding. Indeed, an oligonucleotidedependent hammerhead ribozyme (construct MzL/R; Figure 1d) has been developed for which a specific mRNA serves as both effector and substrate [28••]. This allosteric ribozyme has been demonstrated to inhibit the expression
Allosteric ribozymes also provide an opportunity to examine molecular recognition and structural dynamism in nucleic acid structures. Already, the mechanistic function of certain communication modules has been proposed to involve specific local base-pairing rearrangements in response to effector binding [33••]. Furthermore, the evolution of ligand-binding specificities in aptamers that bind theophylline or 3-methylxanthine has revealed the intricacies of molecular recognition in these related aptamer motifs [34••]. The further development of novel allosteric ribozymes will undoubtedly offer additional opportunities to examine molecular recognition and structural dynamism in nucleic acid structure.
Conclusions The development of allosteric nucleic acid catalysts has been accelerated by the intersection of modular rational design principles and combinatorial selection strategies. Engineering allosteric ribozymes that respond to specific effector stimuli will probably become a routine enterprise for creating potential therapeutic agents or molecular switches that are controlled by specific chemical or physical signals. These molecular switches could find specific application in the artificial control of gene expression in cells. Additionally, allosteric ribozymes may find utility as sensors for the detection of virtually any target compound or signal. These prospects for allosteric nucleic acid catalysts will be met with anticipation, as future endeavors apply the recent advances that have been made in allosteric ribozyme engineering.
Update The L1 ligase ribozyme [29••] has recently been engineered to respond to other molecular effectors, such as FMN and theophylline [45]. This study further demonstrates that modular rational design, communication module selection and aptamer domain swapping can be generally applied to the development of allosteric nucleic acid catalysts.
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Nucleic acids
Acknowledgements We thank GAM Emilsson for critical reading of the manuscript and other members of the Breaker laboratory for helpful discussions. This work was supported by research grants from the National Institutes of Health (GM559343), DARPA and the Yale Diabetes Endocrine Research Center (DERC). Support is also provided by fellowships to RRB from the Hellman Family and from the David and Lucile Packard Foundation.
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