Cloning of random-sequence oligodeoxynucleotides

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.     

Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks.

DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues. Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10. Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II. Oligo(dG)10 did not inhibit other activities such as restriction enzymes. Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme. These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge. Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets. The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.     

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

Immobilization of DNA on glassy carbon electrodes for the development of affinity biosensors

The adsorption and electrooxidation of nucleic acids on glassy carbon electrodes are evaluated by using chronopotentiometric stripping analysis. The influence of electrochemical pretreatments, supporting electrolyte, halides and monovalent cations levels as well as the role of the oligonucleotide length and composition, accumulation potential and time on the adsorption and further electrooxidation of oligo(dG)(11) and oligo(dG)(21) are discussed. The adsorption behavior of single and double stranded calf thymus DNA on untreated glassy carbon electrodes is also evaluated. Trace (microg/l) levels of the oligonucleotides and polynucleotides can be readily detected following short accumulation periods with detection limits of 25, 60, 126 and 219 microg/l for oligo(dG)(21), oligo(dG)(11), ss and ds calf thymus DNA, respectively. The confined DNA layers demonstrated to be stable in air, in 0.200 M acetate buffer pH 5.00 and in 0.020 M phosphate buffer pH 7.40+0.50 M NaCl.     

p53-mediated DNA renaturation can mimic strand exchange.

The process of strand exchange is considered to be the hallmark of DNA recombination. Proteins known to carry out such exchange are believed to operate via one or the other of two mechanisms. RecA-like proteins promote the formation of a three-stranded or triplex synaptic intermediate in which strand exchange occurs, whereas other proteins would allow the coordinated exonucleolytic degradation of one strand in the duplex DNA and its replacement by an invading strand of similar sequence and polarity. In view of properties ascribed to it, we have attempted to determine whether p53 belongs to one or the other of these groups of proteins. The in vitro assay used relies on a double-stranded (ds) oligonucleotide (oligo 1+2) and a single-stranded (ss) oligonucleotide (oligo 3), part of which is complementary to oligo 1. The data collected suggest that, under the conditions of the assay, oligo 1+2 undergoes partial denaturation; p53 then catalyzes renaturation of oligo 1 with oligo 3, rather than true strand exchange. Since p53 is not known for being able to 'melt' DNA, it would seem unlikely that this protein would effect strand exchange in vivo without assistance from another, denaturing, protein.     

The histone core exerts a dominant constraint on the structure of DNA in a nucleosome.

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.     

Identification of novel DNA forms in tomato golden mosaic virus infected tissue. Evidence for a two component viral genome.

Extracts obtained from cells infected with the geminivirus tomato golden mosaic (TGMV) are shown to contain, in addition to viral single-stranded DNA, several novel species of virus-specific single- and double- stranded DNA (ss and ds DNA). The results of nuclease studies and electron microscopy suggest that three of the intracellular DNAs are unit-genome length duplexes of closed circular, relaxed circular, and linear form. The remaining ds DNA species are of high molecular weight and appear to be concatamers consisting of two or more unit-length circular ds TGMV DNA resulted in fragments whose combined size is twice the unit-genome length. Thus ds TGMV is composed of two components of nearly identical size but different nucleotide sequence.     

Complementary addressed modification of plasmid DNA

The possibility to accomplish the sequence-specific chemical modification of superhelical DNA with reactive oligonucleotide derivatives was demonstrated. Plasmids containing fragments of the immunoglobulin gene were modified with alkylating derivatives of oligonucleotides complementary to a nucleotide sequence in the immunoglobulin gene. In contrast to the relaxed plasmid DNAs, superhelical DNAs (sigma = -0.1) were found to be attacked by the derivatives at the target nucleotide sequence. The efficiency of the reaction increases with the increase of the plasmids negative superhelicity. It was found also that the denatured derivatives. The sequence-specific modification of plasmid DNAs with the reactive oligonucleotide derivatives can be used for the site-directed mutagenesis and the investigation of the repair processes.     

Preannealing of poly(A) template and oligo(dT) primer is not required for hepatitis C virus RNA-dependent RNA polymerase activity

Preannealed homopolymeric DNAs or RNAs are often used as templates and/or primers to characterize activities of DNA or RNA-dependent RNA polymerases. Based on the calculated melting temperatures (T_m values), however, poly(A)/oligo(dT_12–18) is not expected to form stable duplexes. To determine this, we compared the enzymatic activity of hepatitis C virus polymerase using poly(A)/oligo(dT₁₂) that were or were not preannealed. No significant differences were observed. These results suggest that it is not necessary to perform preannealing reactions for poly(A) and oligo(dT₁₂), making it possible to characterize mechanism of inhibition of NS5B inhibitors against either template RNA poly(A) or primer oligo(dT₁₂) independently.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Synthesis of oligo(ethylene glycol) substituted phosphatidylcholines: Secretory PLA2-targeted precursors of NSAID prodrugs

A series of new phosphatidylcholine analogues with structurally modified sn-2-substituents have been prepared. The synthetic compounds include oligo(ethylene glycol) derivatives with chain-terminal pharmacophores that upon catalytic hydrolysis by phospholipase A₂ yielded a series of oligo(ethylene glycol)-conjugates of the respective drugs. The approach here outlined may open a new way to employ OEG derivatives of phospholipids for therapeutic applications as secretory PLA₂-targeted precursors of prodrugs.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

The interactions of the separated strands of satellite DNAs with other DNAs: 1. Conditions for associations of the alpha-satellite of the guinea pig with heterologous double-stranded DNAs.

The separated H- and L-strands of the alpha-satellite of the guinea pig, Cavea porcellus, recovered from centrifugation in alkaline CsC1 gradients, from complexes with 7 different double-stranded (ds) DNAs including those of 1 bacteriophage, 2 prokaryotes, 2 invertebrates and 2 mammals. The complexes are not artifacts due to in vitro labeling of the satellite, methods of collection, the presence of divalent cations, or the fact that trace amounts of single-stranded (ss) DNAs are used. More complex dsDNAs, such as that recovered from nicked RF M13, do not associate with dsDNAs.     

Modification of Oligo (Poly) Nucleotide Phosphomonoester Groups in Aqueous Solutions

Abstract

Selective modification of oligo (poly) nucleotide phosphomonoester groups in an aqueous medium by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in the presence of various nucleophilic agents has been investigated. Optimal conditions of the modification by amino- and hydroxycompounds have been found. Based on these studies a general efficient method for preparation of oligo (poly) nucleotide phosphoamidates and phosphodiesters in an aqueous solution has been developed. The method allows to prepare both oligodeoxyribonucleotide derivatives at 3′- and 5′-terminal phosphate groups and oligoribonucleotide derivatives at 5′-terminal phosphate groups with 80–100% yields.