An RNA aptamer that shifts the reduction potential of metabolic cofactors.

The discovery of ribozymes has inspired exploration of RNA's potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme's catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by -40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π-π and donor atom-π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

Isolation of DNA aptamers for herbicides under varying divalent metal ion concentrations.

Functional nucleic acids, including aptamers and deoxyribozymes, have become important in a variety of applications, particularly sensors. Aptamers are useful for recognition because of their ability to bind to targets with high selectivity and affinity. They can also be paired with deoxyribozymes to form signaling aptazymes. These aptamers and aptazymes have the potential to significantly improve the detection of small molecule pollutants, such as herbicides, in the environment. One challenge when developing aptazymes is that aptamer selection conditions can vary greatly from optimal deoxyribozyme reaction conditions. Aptamer selections commonly mimic physiological conditions, while deoxyribozyme selections are conducted under a wider range of divalent metal ion conditions. Isolating aptamers under conditions that match deoxyribozyme reaction conditions should ease the development of aptazymes and facilitate the activities of both the binding and catalytic components. Therefore, we conducted in vitro selections under different divalent metal ion conditions to identify DNA aptamers for the herbicides atrazine and alachlor. Conditions were chosen based on optimal reaction conditions for commonly-used deoxyribozymes. Each set of conditions yielded aptamers that were unrelated to aptamers identified under other selection conditions. No particular set of conditions stood out as being optimal from initial binding analysis. The best aptamers bound their target with high-micromolar to low-millimolar affinity, similar to the concentrations used during the selection procedures, as well as regulatory guidelines. Our results demonstrate that different metal ion concentrations can achieve the common goal of binding to a particular target, while providing aptamers that function under alternate conditions.

Effect of Aptamer Binding on the Electron-Transfer Properties of Redox Cofactors.

In vitro selection or SELEX has allowed for the identification of functional nucleic acids (FNAs) that can potentially mimic and replace protein enzymes. These FNAs likely interact with cofactors, just like enzymes bind cofactors in their active sites. Investigating how FNA binding affects cofactor properties is important for understanding how an active site is formed and for developing useful enzyme mimics. Oxidoreductase enzymes contain cofactors in their active sites that allow the enzymes to do redox chemistry. In certain applications, these redox cofactors act as electron-transfer shuttles that transport electrons between the enzymes' active sites and electrode surfaces. Three redox cofactors commonly found in oxidoreductases are flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD(+)), and pyrroloquinoline quinone (PQQ). We are interested in investigating how DNA aptamers that bind these cofactors influence the cofactors' redox abilities and if these aptamer-cofactor complexes could serve as redox catalysts. We employed cyclic voltammetry and amperometry to study the electrochemical properties of NAD(+) and PQQ when bound to DNA aptamers. Our results suggest that the aptamers provide a stable environment for the cofactor to participate in redox reactions, although enhanced redox activity was not observed. This work provides a foundation for the development of new FNAs capable of redox activity.

Enhanced deoxyribozyme-catalyzed RNA ligation in the presence of organic cosolvents.

Deoxyribozyme and aptamer selections are typically conducted in aqueous buffer solutions. Using nonaqueous cosolvents in selection experiments will help expand the activity of deoxyribozymes with non-oligonucleotide substrates and will allow identification of new aptamers for nonprotein targets. We undertook in vitro selections utilizing a small amount of methanol in the reaction to keep the herbicides alachlor and atrazine in solution with the goal of identifying deoxyribozymes that require these herbicides for activity. The resulting deoxyribozymes successfully catalyze RNA ligation, but do not require alachlor or atrazine. Surprisingly, some of these deoxyribozymes displayed better catalytic activity in the presence of methanol over just aqueous buffer. We investigated several organic cosolvents to see if this enhancement was limited to methanol and found that other cosolvents, including ethanol, DMSO, and DMF, supported activity; in some cases, greater enhancement was observed. On the basis of these results, we tested two other previously identified RNA-ligating deoxyribozymes to assess their tolerance of cosolvents and determined that different deoxyribozymes showed different responses to the cosolvents. Our results demonstrate that deoxyribozymes can tolerate and, in some cases, display enhanced activity in alternative solvent conditions. These findings will facilitate the development of responsive deoxyribozyme systems utilizing components with limited water solubility.

Investigation of a deoxyribozyme as a biofuel cell catalyst.

We examined the ability of a previously identified peroxidase deoxyribozyme to be employed as a catalyst in biofuel cells, as a possible replacement for oxidoreductase proteins. We constructed a biocathode using a covalently linked version of the peroxidase deoxyribozyme-hemin complex and successfully paired it with a glucose dehydrogenase-modified bioanode for power production.

Modulation of an RNA-branching deoxyribozyme by a small molecule.

We have engineered an RNA-branching deoxyribozyme to respond positively to ATP, resulting in modulated control of ligation activity that may be applicable to sensor and nanotechnology applications.

Use of deoxyribozymes in RNA research.

Since their first identification by in vitro selection in 1994, deoxyribozymes have been developed to catalyze a variety of chemical reactions. The first DNA-catalyzed reaction was cleavage of a ribonucleotide linkage within an oligonucleotide substrate. In subsequent years, growing collections of deoxyribozymes have been developed for several reactions that have practical utility for RNA research. These deoxyribozymes are useful for site-specific RNA cleavage as well as ligation to form linear, branched, and lariat RNA products. An application related to RNA ligation is deoxyribozyme-catalyzed labeling of RNA (DECAL), which is used to attach a biophysical tag to a desired RNA sequence at a specific position. With current achievements and likely future developments, deoxyribozymes are a useful contributor to the toolbox of RNA research methods.

Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand.

DNA with a 5'-adenylpyrophosphoryl cap (5'-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5'-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5'-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5'-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates.

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.

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.

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.

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.