Group I aptazymes as genetic regulatory switches
© Thompson et al; licensee BioMed Central Ltd. 2002
Received: 13 June 2002
Accepted: 4 December 2002
Published: 4 December 2002
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 self-splicing 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.
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.
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.
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 . 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 . 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  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 theophylline . 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 .
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
Activation in vitro
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.
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 ΔP6-6; 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 . Defects that poise a ribozyme between active and inactive conformers have previously been used to engineer effector-dependence .
k1 (per hour)
Based on these results, the primary mechanism of activation is likely the same as has been observed for other aptazymes: ligand-induced conformational changes that stabilize functional ribozyme sequences and structures. However, the Group I self-splicing intron is a much more complicated ribozyme than either the hammerhead or the L1 ligase; for example, the tertiary structure of the Group I intron is established by a complicated folding pathway. [22–26] Therefore, it was possible that theophylline-binding influenced the overall folding pathway of the engineered Group I aptazyme. In order to assess this possibility we examined the theophylline-dependence of Th2P6 splicing reactions in vitro following prolonged incubation (30 minutes) to allow for re-folding. No significant change in the extent of ligand-dependent activation was observed following pre-incubation (data not shown). These results argue that both GTP and theophylline may need to be present to influence the folding pathway of the engineered Group I aptazymes. These results are also consistent with previous physical and kinetic [28–30] data that shows that the folding of the Group I intron is dependent in part upon GTP.
Gene regulation in vivo
Several discrepancies were noted between intron activation in vitro and effector-dependent growth. This is not surprising; other studies have revealed that there is often little correlation between ribozyme activity in vitro and in vivo . However, since it is known that relatively small changes in gene expression can greatly affect the metabolism of an organism, another possible explanation for the apparent discrepancies is that relatively small modulations in intron splicing are amplified via cellular metabolism to yield growth phenotypes. This would seem especially true in the current set of experiments, since the modulation of Thymidylate Synthase expression is known to greatly affect thymidine levels and cell growth . For example, E. coli that are starved for thymine quickly die, a phenomenon known as 'thymineless death' . In this respect, Group I aptazymes that may have yielded lower amounts of functional Thymidylate Synthase (e.g., Th3P6, which was less active than Th2P6; or Th1P6, which mis-spliced) did not allow bacterial growth. In contrast, aptazymes that may have yielded even modestly greater amounts of functional Thymidylate Synthase (e.g., Th1P5, which was both highly active and modestly activated in vitro) allowed bacterial growth.
The ability to engineer Group I introns to be responsive to effector molecules has numerous potential applications. For example, it may prove possible to develop new gene therapies in which patients rely upon drugs that can differentially activate gene expression, rather than having to rely upon a set level of endogenous expression of an introduced gene. Similarly, it may prove possible to use effector-dependent splicing to more finely monitor gene expression in vivo. A drug could be localized to particular organs, cells, or organelles, and splicing of the aptazyme could be monitored via a reporter gene such as luciferase. It is also possible to imagine that engineered introns introduced into reporter genes may find application in high-throughput, cell-based screening assays that monitor drug uptake or efficacy. These strategies should prove useful not only for self-splicing introns activated by drugs, but for introns activated by proteins or peptides. In this regard, it is notable that some natural Group I introns have proven to be highly dependent upon protein cofactors, and that Group I introns have been engineered whose tertiary structure and activity is dependent upon a peptide . Such applications could also be melded with other innovations dependent upon the Group I self-splicing intron, such as the introduction of a trans-splicing intron that can potentially repair or modulate the expression of a given mRNA. [37–39]
Group I aptazymes that were dependent upon theophylline both in vitro and in vivo were engineered by conjoining the thymidylate synthase (td) intron with an anti-theophylline aptamer. Although the in vitro activation of splicing was modest (up to an 18-fold change in initial rate), this change was more than enough to mediate the survival of a thymidine-dependent E. coli strain. The ligand specificity of the anti-theophylline aptamer carried over to the theophylline-dependent aptazyme, and no activation was seen with caffeine, which differed by a single methyl group from theophylline. The effector specificity could be rationally changed by changing the specificity of the conjoined aptamer: when a mutation was introduced into the anti-theophylline aptamer that led to discrimination against theophylline and in favor of 3-methylxanthine (that also differed from theophylline by a single methyl group), the aptamer became solely responsive to 3-methylxanthine in vivo. Group I aptazymes may have a number of important biotechnology applications, including use as in vivo, real-time reporters and as regulatable gene therapeutics.
E. colistrains and growth media
E. coli strain C600ThyA::KanR was used for the plate assays and in vivo growth curves. INVaF' (Invitrogen, Carlsbad, CA) was used for cloning and plasmid amplification. Bacterial starter cultures were grown in LB supplemented with thymine (50 mg/l). Screening for thymidine auxotrophy was done in minimal media supplemented with 0.1% Norit A-treated casamino acids (MM) and MM supplemented with thymine (50 mg/l) (MMT). Plates contained Bacto agar (1.5%). Ampicillin (50 mg/l) and kanamycin (100 mg/l) were added to all growth media.
Construction of the tdintron aptazymes
The wild type plasmid pTZtd1304  contains a fully functional 265 nt derivative of the 1016 nt wild type intron . This derivative also contains the substitutions U34A, which introduces a SpeI site, and U976G, which introduces an EcoRI site. The constructs were made using standard solid phase DNA synthesis, then were PCR-amplified and cloned into pTZtd1304. The parental P6 aptazyme construct was generated by two overlapping oligos, Gp1Wt2 Gp1Wt2.122 (GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TTG TAG GAC TGC CCG GGT TCT ACA TAA ATG CCT AAC GAC TAT CCC TT) and Gp1Wt3.129 (TAA TCT TAC CCC GGA ATT ATA TCC AGC TGC ATG TCA CCA TGC AGA GCA GAC TAT ATC TCC AAC TTG TTA AAG CAA GTT GTC TAT CGT TTC GAG TCA CTT GAC CCT ACT CCC CAA AGG GAT AGT CGT TAG). These oligonucleotides (100 pmole) were annealed and extended with AMV reverse transcriptase (Amersham Pharmacia Biotech, Piscataway, NJ; 45 units) in AMV RT buffer (50 mM Tris-HCl, pH 8.3, 8 mM MgCl2, 50 mM NaCl, 1 mM DTT) and dNTPS (200 μM) for 30 minutes at 37°C. The resulting double-stranded DNA was diluted 1:50 and amplified using primers SpeI.24 (TTA TAC TAG TAA TCT ATC TAA ACG; 0.4 μM) and EcoRI.24 (CCC GGA ATT CTA TCC AGC TGC ATG; 0.4 μM) in PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.005% gelatin), dNTPS (200 μM) and Taq DNA polymerase (Promega, Madison, WI; 1.5 units). The reactions were thermocycled 15 times at 94°C for 30 seconds, 45°C for 30 seconds, 72°C for 1 minute and then purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA). The PCR product was digested with SpeI (New England Biolabs, Beverly, MA; 20 units) and EcoRI (50 units) in buffer (50 mM NaCl, 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.025% Triton X-100, 100 μg/ml BSA) at 37°C for 60 minutes, purified, and cloned into SpeI/EcoRI digested pTZtd1304. The negative control and aptazyme constructs were made as described except that Gp1Wt3.129 was replaced with oligonucleotides of the appropriate sequence: B11 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TGC CTA ACG ACT ATC CCT T, Th1P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TTA TAC CAG CAT CGT CTT GAT GCC CTT GGC AGA TAA ATG CCT AAC GAC TAT CCC TT, Th2P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TTG ATA CCA GCA TCG TCT TGA TGC CCT TGG CAG CAT AAA TGC CTA ACG ACT ATC CCT T, Th3P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCA TAC CAG CAT CGT CTT GAT GCC CTT GGC AGG CCT AAC GAC TAT CCC TT, Th4P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TAT ACC AGC ATC GTC TTG ATG CCC TTG GCA GTA AAT GCC TAA CGA CTA TCC CTT, Th5P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT ATA CCA GCA TCG TCT TGA TGC CCT TGG CAG CTA ACG ACT ATC CCT T, Th6P6 GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GAT ACC AGC ATC GTC TTG ATG CCC TTG GCA GCC TAA CGA CTA TCC CTT, Th1P5 TGA GTA TAA GGT GAC TTA TAC TAG TAA TCT ATC TAA ACG GGG AAC CTC TAT ACC AGC ATC GTC TTG ATG CCC TTG GCA GAG ACA ATC CCG TGC TAA ATT GTA GGA CTG CCC GGG TTC TAC ATA AAT GCC TAA CGA CTA TCC CTT, Th2P5 TGA GTA TAA GGT GAC TTA TAC TAG TAA TCT ATC TAA ACG GGG AAC CTA TAC CAG CAT CGT CTT GAT GCC CTT GGC AGA CAA TCC CGT GCT AAA TTG TAG GAC TGC CCG GGT TCT ACA TAA ATG CCT AAC GAC TAT CCC TT, 3Mex2P6 GTA ATC TAT CTA AAC GGG GAA CCT CTC TAG TAG ACA ATC CCG TGC TAA ATT GAT ACC AGC ATC GTC TTG ATG CCA TTG GCA GCA TAA ATG CCT AAC GAC TAT CCC TT, and Th2P6.D GTA ATC TAT CTA AAC GGG GAA CCT CTC TAG TAG ACA ATC CCG TGC TAA ATT GAT ACC AGC ATC GTC TTG ATG CCC TTG GTT GCA TAA ATG CCT AAC GAC TAT CCC TT.
The introns were PCR-amplified with KM37 (GAT AAT ACG ACT CAC TAT AAT GGC ATT ACC GCC TTG T) and GM24 (GCT CTA GAC TTA GCT ACA ATA TGA AC) in 25 μl reactions under the conditions stated above and cycled 20 times. A portion of the reaction (5 μl) was run on a 3% agarose gel and the PCR product band was stabbed with a pipette tip. The agarose plug was added to a fresh PCR reaction (100 μl) and cycled 15 times; DNA was purified using a QIAquick kit and quantitated. The PCR product (2 μg in 50 μl) was added to an in vitro transcription reaction containing Ampliscribe T7 RNA polymerase (Epicentre), RNase inhibitor (GIBCO BRL, Rockville, MD; 5 units), low Mg2+ buffer (30 mM Tris-HCl, pH 8, 7.5 mM DTT, 4.5 mM MgCl2, 1.5 mM spermidine), UTP (1.25 mM), ATP (2.5 mM), GTP (2.5 mM), CTP (7.5 mM) and α-32P UTP (NEN, Boston, MA; 20 μCi; 3000 mCi/mmol), and incubated at 37°C for 2 hours. DNase (GIBCO BRL, 295 units) was added and the reaction was incubated at 37°C for an additional 30 minutes. The RNA was purified using Centri-Sep columns (Princeton Separations, Adelphia, NJ) and quantitated.
In vitrosplicing assays
The assays were performed by heating the RNA (500 nM) in H2O to 70°C for 3 minutes then transferring to ice for 1 minute. Splicing buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 3 mM MgCl2), effector (theophylline, 1.5 mM) was added and the reactions were incubated on ice for an additional 15 minutes. At this time a 4.5 μl aliquot was removed for a zero time point and quenched with 5 μl stop dye (95% formamide, 20 mM EDTA, 0.5% xylene cyanol, and 0.5% bromophenol blue). GTP (50 μM) was added to the remaining reaction (5 μl total volume) to start the splicing reaction. The reaction was incubated at 37°C for 30 minutes and then terminated with stop dye (5 μl). The reactions were heated to 70°C for 3 minutes and 5 μl was analyzed on a 8% denaturing polyacrylamide gel. The gel was dried, exposed to a phosphor screen and analyzed using a Molecular Dynamics Phosphorimager (Sunnyvale, CA).
The reaction volumes were increased for the rate determination assay. Aliquots were taken at intervals between 0 minutes and 15 minutes and terminated with stop dye. The reactions were analyzed as described above. For each time point, the number of counts in unreacted intron, spliced intron, and circularized intron were determined. Background counts were subtracted from these values. The fraction of reacted intron was then calculated as:
(circular intron + spliced intron) / (unreacted intron + circular intron + spliced intron)
The individual time points were averaged together, the fraction reacted was plotted versus time, and rate constants were extracted by fitting the data to a single-exponential equation:
F = A * e (-kt)
The plotted data and rate constants for Th1P5 and Th2P6 constructs are shown in Figures 2b and 2c and Table 2. The changes in the extent of reaction observed between individual experiments, and thus the attendant error bars, were consistent with those observed during the kinetic analyses of other ribozymes. For example, studies of the hairpin ribozyme have shown that data points can vary between individual experiments by as much as 50% . Our data points typically varied from one another by less than 30%.
In vivoplate assay
The plasmids containing the various group I constructs were transformed into competent C600ThyA::KanR cells and grown in LB with kanamycin overnight. A small aliquot (3 μl) of overnight cell culture was mixed with effector (theophylline, 7.5 mM) or H2O, spotted on plates, and grown overnight at 37°C. As a positive control, all constructs were also plated on minimal media plates with thymine (MMT) and assayed for viability.
In vivogrowth curves
Cells grown overnight in LB were diluted 1:100 in MM containing either theophylline, caffeine, 3-methylxanthine or no effector, and analyzed on a Microbiology Workstation Bioscreen C (Labsystems, Inc., Franklin, MA).
RNA was isolated from an overnight culture using a MasterPure RNA purification kit (Epicentre, Madison, WI) and amplified by RT-PCR using primers KM37 and GM24 following the protocol provided for Tth polymerase. The products were separated and analyzed on a 3% agarose gel.
This work was funded by the Office of Naval Research (Grant #: N00014-99-1-0861), the Defense Advanced Research Projects Agency (Grant #: DAAD19-99-1-0207), and the National Institutes of Health (Grant #: 1R01 GM61789-01). We would like to thank Ken Johnson for assistance with kinetic analyses.
- Peracchi A, Beigelman L, Usman N, Herschlag D: Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases. Proc Natl Acad Sci U S A. 1996, 93 (21): 11522-11527. 10.1073/pnas.93.21.11522.View ArticleGoogle Scholar
- Porta H, Lizardi PM: An allosteric hammerhead ribozyme. Biotechnology (N Y). 1995, 13 (2): 161-164. 10.1038/nbt0295-161.View ArticleGoogle Scholar
- Hermann T, Patel DJ: Adaptive recognition by nucleic acid aptamers. Science. 2000, 287 (5454): 820-825. 10.1126/science.287.5454.820.View ArticleGoogle Scholar
- Jiang F, Gorin A, Hu W, Majumdar A, Baskerville S, Xu W, Ellington A, Patel DJ: Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples. Structure Fold Des. 1999, 7 (12): 1461-1472. 10.1016/S0969-2126(00)88337-9.View ArticleGoogle Scholar
- 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 (5): 645-664. 10.1006/jmbi.1997.1281.View ArticleGoogle Scholar
- Patel DJ: Structural analysis of nucleic acid aptamers. Curr Opin Chem Biol. 1997, 1 (1): 32-46. 10.1016/S1367-5931(97)80106-8.View ArticleGoogle Scholar
- Soukup GA, Breaker RR: Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization. Structure Fold Des. 1999, 7 (7): 783-791. 10.1016/S0969-2126(99)80102-6.View ArticleGoogle Scholar
- Soukup GA, Breaker RR: Nucleic acid molecular switches. Trends Biotechnol. 1999, 17 (12): 469-476. 10.1016/S0167-7799(99)01383-9.View ArticleGoogle Scholar
- Marshall KA, Ellington AD: Training ribozymes to switch. Nat Struct Biol. 1999, 6 (11): 992-994. 10.1038/14872.View ArticleGoogle Scholar
- Jenison RD, Gill SC, Pardi A, Polisky B: High-resolution molecular discrimination by RNA. Science. 1994, 263 (5152): 1425-1429.View ArticleGoogle Scholar
- Zimmermann GR, Shields TP, Jenison RD, Wick CL, Pardi A: A semiconserved residue inhibits complex formation by stabilizing interactions in the free state of a theophylline-binding RNA. Biochemistry. 1998, 37 (25): 9186-9192. 10.1021/bi980082s.View ArticleGoogle Scholar
- Zimmermann GR, Jenison RD, Wick CL, Simorre JP, Pardi A: Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA [see comments]. Nat Struct Biol. 1997, 4 (8): 644-649. 10.1038/nsb0897-644.View ArticleGoogle Scholar
- Soukup GA, Breaker RR: Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A. 1999, 96 (7): 3584-3589. 10.1073/pnas.96.7.3584.View ArticleGoogle Scholar
- Robertson MP, Ellington AD: Design and optimization of effector-activated ribozyme ligases. Nucleic Acids Res. 2000, 28 (8): 1751-1759. 10.1093/nar/28.8.1751.View ArticleGoogle Scholar
- Galloway Salvo JL, Coetzee T, Belfort M: Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region. J Mol Biol. 1990, 211 (3): 537-549. 10.1016/0022-2836(90)90264-M.View ArticleGoogle Scholar
- Mohr G, Zhang A, Gianelos JA, Belfort M, Lambowitz AM: The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core. Cell. 1992, 69 (3): 483-494. 10.1016/0092-8674(92)90449-M.View ArticleGoogle Scholar
- Myers CA, Wallweber GJ, Rennard R, Kemel Y, Caprara MG, Mohr G, Lambowitz AM: A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core. J Mol Biol. 1996, 262 (2): 87-104. 10.1006/jmbi.1996.0501.View ArticleGoogle Scholar
- Joyce GF, van der Horst G, Inoue T: Catalytic activity is retained in the Tetrahymena group I intron despite removal of the large extension of element P5. Nucleic Acids Res. 1989, 17 (19): 7879-7889.View ArticleGoogle Scholar
- Mohr G, Caprara MG, Guo Q, Lambowitz AM: A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme [see comments]. Nature. 1994, 370 (6485): 147-150. 10.1038/370147a0.View ArticleGoogle Scholar
- Soukup GA, Breaker RR: Allosteric nucleic acid catalysts. Curr Opin Struct Biol. 2000, 10 (3): 318-25. 10.1016/S0959-440X(00)00090-7.View ArticleGoogle Scholar
- Tang J, Breaker RR: Rational design of allosteric ribozymes. Chem Biol. 1997, 4 (6): 453-459. 10.1016/S1074-5521(97)90197-6.View ArticleGoogle Scholar
- Treiber DK, Williamson JR: Exposing the kinetic traps in RNA folding. Curr Opin Struct Biol. 1999, 9 (3): 339-345. 10.1016/S0959-440X(99)80045-1.View ArticleGoogle Scholar
- Treiber DK, Rook MS, Zarrinkar PP, Williamson JR: Kinetic intermediates trapped by native interactions in RNA folding. Science. 1998, 279 (5358): 1943-1946. 10.1126/science.279.5358.1943.View ArticleGoogle Scholar
- Russell R, Herschlag D: New pathways in folding of the Tetrahymena group I RNA enzyme. J Mol Biol. 1999, 291 (5): 1155-1167. 10.1006/jmbi.1999.3026.View ArticleGoogle Scholar
- Wu M, Tinoco I: RNA folding causes secondary structure rearrangement. Proc Natl Acad Sci U S A. 1998, 95 (20): 11555-11560. 10.1073/pnas.95.20.11555.View ArticleGoogle Scholar
- Doherty EA, Doudna JA: The P4-P6 domain directs higher order folding of the Tetrahymena ribozyme core. Biochemistry. 1997, 36 (11): 3159-3169. 10.1021/bi962428+.View ArticleGoogle Scholar
- Emerick VL, Woodson SA: Fingerprinting the folding of a group I precursor RNA. Proc Natl Acad Sci U S A. 1994, 91 (21): 9675-9679.View ArticleGoogle Scholar
- Bevilacqua PC, Johnson KA, Turner DH: Cooperative and anticooperative binding to a ribozyme. Proc Natl Acad Sci U S A. 1993, 90 (18): 8357-8361.View ArticleGoogle Scholar
- Bevilacqua PC, Li Y, Turner DH: Fluorescence-detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5' cleavage site into a high free energy binding mode in the Tetrahymena ribozyme. Biochemistry. 1994, 33 (37): 11340-11348. 10.1021/bi00203a032.View ArticleGoogle Scholar
- McConnell TS, Cech TR, Herschlag D: Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding. Proc Natl Acad Sci U S A. 1993, 90 (18): 8362-8366.View ArticleGoogle Scholar
- Kato Y, Kuwabara T, Warashina M, Toda H, Taira K: Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells. J Biol Chem. 2001, 276 (18): 15378-15385. 10.1074/jbc.M010570200.View ArticleGoogle Scholar
- Crecy-Lagard de, Bellalou VA J, Mutzel R, Marliere P: Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli. BMC Biotechnol. 2001, 1 (1): 10-10.1186/1472-6750-1-10.View ArticleGoogle Scholar
- Ahmad SI, Kirk SH, Eisenstark A: Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu Rev Microbiol. 1998, 52: 591-625. 10.1146/annurev.micro.52.1.591.View ArticleGoogle Scholar
- Soukup GA, Emilsson GA, Breaker RR: Altering molecular recognition of RNA aptamers by allosteric selection. J Mol Biol. 2000, 298 (4): 623-632. 10.1006/jmbi.2000.3704.View ArticleGoogle Scholar
- Lambowitz AM, Caprara MG, Zimmerly S, Perlman PS: Group I and group II ribozymes as RNPs: clues to the past and guides to the future. in The RNA World, C.T.R. Edited by: Gesteland RF, Atkins JF. 1999, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 451-485.Google Scholar
- Atsumi S, Ikawa Y, Shiraishi H, Inoue T: Design and development of a catalytic ribonucleoprotein. Embo J. 2001, 20 (19): 5453-5460. 10.1093/emboj/20.19.5453.View ArticleGoogle Scholar
- Watanabe T, Sullenger BA: RNA repair: a novel approach to gene therapy. Adv Drug Deliv Rev. 2000, 44: 2-3. 10.1016/S0169-409X(00)00089-2.View ArticleGoogle Scholar
- Watanabe T, Sullenger BA: Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc Natl Acad Sci U S A. 2000, 97 (15): 8490-8494. 10.1073/pnas.150104097.View ArticleGoogle Scholar
- Sullenger BA, Cech TR: Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing. Nature. 1994, 371 (6498): 619-622. 10.1038/371619a0.View ArticleGoogle Scholar
- Esteban JA, Walter NG, Kotzorek G, Heckman JE, Burke JM: Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme. Proc Natl Acad Sci U S A. 1998, 95 (11): 6091-6096. 10.1073/pnas.95.11.6091.View ArticleGoogle Scholar
- Damberger SH, Gutell RR: A Comparative Database of Group I Intron Structures. Nucleic Acids Res. 1994, 22: 3508-3510.View ArticleGoogle Scholar
- Cech TR, Damberger SH, Gutell RR: Representation of the secondary and tertiary structure of group I introns. Nat Struct Biol. 1994, 1 (5): 273-280. 10.1038/nsb0594-273.View ArticleGoogle Scholar
- Zimmermann GR, Wick CL, Shields TP, Jenison RD, Pardi A: Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer. Rna. 2000, 6 (5): 659-667. 10.1017/S1355838200000169.View ArticleGoogle Scholar
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