Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C(2)H(2)-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.     

Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.     

Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.     

The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates.

A steady-state kinetic analysis of the cleavage of the oligonucleotides d(CGCTTTTTTGC) (d(y)), d(GCAAAAAAGCG) (d(r)), r(CGCUUUUUUGC) (r(y)) and r(GCAAAAAAGCG) (r(r)) in single and double-stranded form by the extracellular Serratia marcescens endonuclease, in conjunction with structural data from a circular dichroism spectroscopic analysis of these substrates, suggests that oligonucleotides adopting the A-conformation are preferred over those adopting the B-conformation as substrates. Relative catalytic efficiencies (kcat/KM) for the cleavage of the homo- and heteroduplexes follow the order r(r).r(y) (1.0)>r(r).d(y) (0.9)>d(r). r(y) (0.7)>d(r).d(y) (0.3). The purine-rich single-stranded oligonucleotides r(r) and d(r), are cleaved more efficiently than the pyrimidine-rich oligonucleotides, r(y) and d(y), presumably because they adopt helical structures with pronounced base stacking. Except for the double-stranded oligodeoxynucleotide substrate, the individual strands are cleaved more efficiently when incorporated into a duplex, than in a single-stranded form. Cleavage experiments with various polynucleotides, including a viroid RNA and a specifically designed 167 bp DNA, confirm that double-stranded A-form nucleic acids are preferentially attacked by Serratia nuclease. In an attempt to analyze the basis of these preferences, we have mutated the amino acid residues Tyr76 and Trp123 of Serratia nuclease. These residues are located close to the active site and are conserved in all members of the Serratia nuclease family, suggesting that they could be involved in substrate binding, e.g. by stacking interactions with the bases, which could lead to the cleavage preferences observed. However, only effects on the activity, but no change of the sequence or substrate preferences, were detected upon substitution of these amino acid residues, ruling out any involvement of these residues in the A-form preference of Serratia nuclease.     

Design and synthesis of metal-free artificial ribonucleases

The present review is aimed at giving a general overview of our results in the field of designing and synthesizing simple peptide-like molecules that mimic structural and functional aspects of natural ribonucleases, as well as designing oligonucleotide-based artificial ribonucleases.     

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.     

Photoinduced DNA cleavage by naphthalimide hydroperoxide-specific strand scission at -GG- site of DNA

We have prepared a series of naphthalene hydroperoxides (1-3) which possess hydroperoxy group at gamma-position of imide carbonyl. Upon photoirradiation (greater than 350 nm) hydroperoxides (1-3) decomposed with efficient generation of hydroxyl radical, which was confirmed by esr spin trapping technique using dimethylpyrroline oxide as a spin trapper. All these hydroperoxides induced DNA strand scission upon photoirradiation (greater than 350 nm), especially hydroperoxide 3 cleaved plasmid phi X 174 DNA (Form I) to give nicked (Form II) and linear (Form III) DNA even at 1 microM concentration. Further, it was observed that 3 exclusively cleaved DNA at the 5'-G site of -GG-sequence.     

Hydrolysis of plasmid DNA catalyzed by Co(III) complex of cyclen attached to polystyrene

Reactivity of the Co(III) complex of cyclen (CoCyc) in the hydrolytic cleavage of supercoiled pUC18 DNA leading to the formation of the corresponding open circular form was enhanced by >200 times upon attachment of CoCyc to cross-linked polystyrenes. Thus, half-lives as short as 40 min were achieved by the resin-based CoCyc in cleavage of the supercoiled DNA at 4 degrees C.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.     

Hydrolysis of DNA by Ce(IV)/EDTA complexes--the mechanism of DNA cleavage and design of artificial restriction enzymes.

Homogeneous Ce(IV) complex of EDTA promptly hydrolyzes oligonucleotides under physiological conditions. Moreover, the activity of Ce(IV)/EDTA for DNA hydrolysis is promoted by the addition of amines. When [Ce(IV)/EDTA] = 5 mumol dm3 and [ethylenediamine] = 100 mmol dm3, the catalytic activity is about 50 times as large as that of Ce(IV)/EDTA. The combination of Ce(IV)/EDTA and amines is eminent tools for the future molecular biology and biotechnology.     

Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

Background  

The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation.     

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Single strand breakage and repair in eukaryotic DNA as assayed by S1 nuclease.

A sensitive new approach for measuring the repair of single strand breaks in DNA induced by low doses of gamma irradiation was tested in cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and in normal cells of early and late passages. The assay is based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation is shown to follow the relation, in F = -(1/Mn - const) - tbeta where F is the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. Mn is the number-average molecular weight of DNA between single strand breaks. beta less than 1 is an empirically determined constant. The results suggest an increase in the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.     

Synthesis of aminoazalactams as cyclic mimetics of basic alkyl amino acids

Novel medium ring conformationaly constrained amino acid derivatives 2 for incorporation into collagenase inhibitors were prepared, utilising natural amino acids as starting materials, via a reductive intramolecular imine cyclisation.     

Monitoring of RNA clearance in a novel plasmid DNA purification process

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.