Biomedical subjects

Stephen M Testa

Publications and source records attributed to Stephen M Testa.

8 recordsLinked to original sources

PubMed2005

Trans insertion-splicing: ribozyme-catalyzed insertion of targeted sequences into RNAs.

A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl(2) for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an omegaG on the ribozyme and an omegaG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.

Catalysis↗

PubMed2005

5' transcript replacement in vitro catalyzed by a group I intron-derived ribozyme.

Group I intron-derived ribozymes can perform a variety of catalytic reactions, including the replacement of the 3' end of a mutant RNA transcript with a corrected version of the transcript [Sullenger, B. A., and Cech, T. R. (1994) Nature 371, 619-622]. We now demonstrate in vitro that a ribozyme, derived from a Pneumocystis carinii group I intron, can replace the 5' end of a targeted exogenous RNA with an endogenous RNA. Our model system is a short synthetic mimic of a k-ras transcript, in which substitution mutations at codon 12 are implicated in a host of cancer types. In these experiments, yields of up to 70% were obtained. We analyzed the length dependence of two molecular contacts, P9.0 and P10, that occur between the ribozyme and the exogenous k-ras mimic, and determined that longer, and thus more stable, interactions result in higher product yields. Furthermore, the length of the loop region L1 can substantially influence the yield and the rate of the reaction. These results are a further demonstration that group I intron-derived ribozymes are quite malleable in terms of intermolecular recognition and catalysis, and that these properties can be exploited in developing potentially useful biochemical tools.

5' Untranslated Regions↗

PubMed2005

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme.

We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this trans excision-splicing ribozyme in a cellular environment, specifically Escherichia coli, was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for trans excision-splicing. We show that the ribozyme is able to catalyze the trans excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.

Escherichia coli↗

PubMed2005

Molecular recognition in a trans excision-splicing ribozyme: non-Watson-Crick base pairs at the 5' splice site and omegaG at the 3' splice site can play a role in determining the binding register of reaction substrates.

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5' splice site and a guanosine in the omega position at the 3' splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5' splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5' cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5'splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5' splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain omegaG, suggesting that omegaG can play a similar role. We report that omegaG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that omegaG is sufficient as a 3' molecular recognition element.

Base Pairing↗

PubMed2004

Enhancing the second step of the trans excision-splicing reaction of a group I ribozyme by exploiting P9.0 and P10 for intermolecular recognition.

We previously reported that a group I intron-derived ribozyme can catalyze the excision of targeted sequences from within RNAs in vitro and that dissociation of the bridge-3' exon intermediate between the two reaction steps is a significant contributing factor to low product yields. We now analyze the effects of increasing the length, and thus the strength, of helices P9.0 and P10, which occur between the ribozyme and the bridge-3' exon region of the substrate, on this trans excision-splicing reaction. Using substrates where lengthy targeted regions are excised, these modifications can significantly increase product yields, specifically by enhancing the second reaction step. A threshold for product formation is obtained, however, at around five base pairs for P10 and eight base pairs for P9.0. Nevertheless, elongating P9.0 appears to be the more effective strategy, as both substrate binding and the rate of the second reaction step increase. In addition, P10 is required when P9.0 is not elongated. Also, a strong P9.0 helix cannot replace a weaker P10 helix, indicating that P9.0 and P10 play somewhat distinct roles in the reaction. We also show that second-step inhibition stems from the formation of an extended P1 helix (P1ex), consisting of as little as a single Watson-Crick base pair, as well as the mere presence of substrate nucleosides immediately downstream from P10. Both of these inhibitory components can be overcome by utilizing P9.0 and P10 elongated ribozymes. This work sets forth an initial framework for rationally designing more effective trans excision-splicing ribozymes.

Animals↗

PubMed2003

Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions.

T4 DNA ligase catalyzes the template-dependent ligation of DNA. Using T4 DNA ligase under specific experimental conditions, we demonstrate that each of the four canonical nucleosides, centrally located on a template molecule such that they flank the site of ligation, can direct the ligation of nucleic acids regardless of the identity of the terminal nucleosides being covalently joined. This universal templating capability extends to those positions adjacent to the ligation junction. This is the first report, irrespective of the ligation method used or the identity of the template nucleosides (including analogs), which shows that nucleosides can act essentially as universal templates at ligation junctions in vitro. The canonical nucleosides do, however, differ in their ability to template sequence- independent ligations, with thymidine and guanosine being equally effective, yet more effective than adenosine and cytidine. Results indicate that hybridization strength surrounding the ligation junction is an important factor. The implications of this previously undiscovered property of T4 DNA ligase with canonical nucleosides are discussed.

Bacteriophage T4↗

PubMed2003

Molecular recognition properties of IGS-mediated reactions catalyzed by a Pneumocystis carinii group I intron.

We report the development, analysis and use of a new combinatorial approach to analyze the substrate sequence dependence of the suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS)-mediated reactions is not high. In addition, the sequence specificity of suicide inhibition decreases with increasing MgCl(2) concentration, reverse cyclization is substantially more sequence specific than suicide inhibition, and multiple reverse cyclization products occur, in part due to the formation of multiple cyclization intermediates. Thermodynamic analysis reveals that a base pair at position -4 of the resultant 5' exon-IGS (P1) helix is crucial for tertiary docking of the P1 helix into the catalytic core of the ribozyme in the suicide inhibition reaction. In contrast to results reported with a Tetrahymena ribozyme, altering the sequence of the IGS of the P.carinii ribozyme can result in a marked reduction in tertiary stability of docking the resultant P1 helix into the catalytic core of the ribozyme. Finally, results indicate that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.

Base Sequence↗

PubMed2002

Ribozyme-catalyzed excision of targeted sequences from within RNAs.

We demonstrate that a group I intron-derived ribozyme from the opportunistic pathogen Pneumocystis carinii can bind an RNA in trans and excise from within it an internal segment, resulting in the splicing of the remaining ends of the RNA back together (the trans excision-splicing reaction). The reaction is intramolecular with regard to substrate. The ribozyme targets its substrate by base pairing with two or three noncontiguous regions on the substrate, and the reaction occurs through a nucleotide cofactor independent mechanism. The excised segment can be as long as 28 nucleotides, or more, and as little as one nucleotide. The potential usefulness of this reaction is demonstrated by engineering a ribozyme that excises the triplet-repeat expansion region from a truncated myotonic dystrophy protein kinase transcript mimic in vitro.

Base Sequence↗