A Base Pair Outside the Catalytic Core of the I‐R3 DNA Enzyme Has a Significant Effect on Its Cleavage Activity: An Improved Catalytic Core Model and an Automated Design Program

Design of nuclease resistant protein kinase cα DNA enzymes with potential therapeutic application

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson-Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson-Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3-R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem-loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson-Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes.

DNA enzymes are RNA-cleaving single stranded DNA molecules. The structure and the catalytic domain of a DNA enzyme were determined by Santro et al. in 1997. In this study, we have designed several types of DNA enzymes (PB2Dz) targeted to the PB2 mRNA translation initiation region of influenza A virus, and examined their cleavage kinetics, nuclease resistance, and a luciferase gene reporter assay. Using a synthetic substrate, these DNA enzymes were shown to have cleavage activity that is dependent on the length of the substrate recognition domain. To confer serum nuclease resistance to the DNA enzymes, we designed a new type of DNA enzyme that has the N3'-P5' phosphoramidate modification (PB2Dz-N) at each terminal. We examined the activity of this DNA enzyme in vivo. The DNA enzymes used in this study inhibited the expression of the PB2-luciferase gene in COS cells. These results suggest that DNA enzymes are potentially useful as gene inactivating agents of influenza A virus.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

A high-throughput analysis of several self-cleaving ribozymes reveals the effect of every possible mutation, and every possible pair of mutations, and how patterns in the relative activity data can be mapped to canonical and non-canonical structural elements.

A high-throughput analysis of several self-cleaving ribozymes reveals the effect of every possible mutation, and every possible pair of mutations, and how patterns in the relative activity data can be mapped to canonical and non-canonical structural elements.

A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells

cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Tumor necrosis factor-alpha (TNF-alpha) probably affects the pathogenesis of heart failure. Here we have investigated the therapeutic potential of a nuclease-resistant DNA enzyme that specifically cleaves TNF-alpha mRNA. A phosphorothioate-modified DNA enzyme was designed to retain similar cleavage activity as its unmodified version, and that inhibited the expression of TNF-alpha in vitro. To test its efficacy in vivo, postinfarction congestive heart failure was induced in anesthetized rats by ligation of the left coronary artery. A 4-wk treatment with the DNA enzyme induced a substantial reduction in left ventricular end-diastolic pressure and lung weight concomitant with an increase in arterial blood pressure and myocardial blood flow compared with controls. The concentration of TNF-alpha in coronary sinus blood was markedly lowered on treatment, and myocardial TNF-alpha mRNA was substantially reduced. Recovery studies showed that the DNA enzyme cleavage activity was present within the myocardium throughout the observation period and had no apparent toxic effects. Our findings indicate that DNA enzyme-based therapy may hold promise in the treatment of this debilitating disease.

The genome of HIV-1 is known to accumulate nucleotide changes throughout the course of disease that result in generation of escape mutants. Therefore, any nucleic acid-based antiviral approach should be targeted against multiple regions of the HIV-1 genome that might significantly delay the appearance of such mutants. We designed several DNA enzymes against the most conserved p24 Gag and the Nef regions in the HIV-1 genome. Sequence-specific cleavage activity was observed for all the DNA enzymes tested. Gag DNA enzyme, which cleaved the target RNA more efficiently in the presence of low levels or physiologic levels of Mg(2+), interfered more effectively with HIV-1 gene expression in virus challenge experiments. The two Nef DNA enzymes, as observed with Gag DNA enzymes, showed significant variation in their cleavage activities in the presence of varying concentration of Mg(2+) and, as expected, did not interfere with the replication of a laboratory-adapted HIV-1 isolate under in vitro culture conditions. The Gag DNA enzymes could be exploited in combination with other promising antiviral approaches.

A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplex-forming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A beta-galactosidase (beta-gal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using beta-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves beta-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave beta-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the beta-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of beta-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.

Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10-23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg(2+) only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.