3′-MODIFIED OLIGO(2′-O-METHYLRIBONUCLEOTIDES) AS IMPROVED PROBES FOR HYBRIDIZATION WITH RNA

A series of octa(2′-O-methylribonucleotides) with an additional 3′-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3′-3′ (“inverted”) bond was synthesized. The exceptional stability of these oligomers to a 3′-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3′-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2′-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal A^mU(T) pair and enhances the mismatch sensitivity.     

Properties of deoxynucleoside 5''-monophosphatase induced by bacteriophage T5 after infection of Escherichia coli.

Bacteriophage T5 induced a deoxynucleoside 5'-monophosphatase during its infection of Escherichia coli. The enzyme was purified about 100-fold. It was clearly distinct from the host 5'-nucleotidase activity in its physical characteristics and substrate specificity. The enzyme was active on deoxynucleoside 5'-monophosphates but was not active as a phosphatase on ribonucleotides, deoxynucleoside 5'-triphosphates, deoxynucleoside 3'-monophosphates, or deoxyoligonucleotides. Furthermore, it did not have oligonucleotidase or exonuclease activity. The enzyme could exist in multimeric form but had a monomer molecular weight of about 25,000.     

Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5′ to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3′ hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

Biochemical studies on the mutagen, 6-N-hydroxylaminopurine. Synthesis of the deoxynucleoside triphosphate and its incorporation into DNA in vitro

The nucleotide analogue, 6-N-hydroxylaminopurine deoxynucleoside triphosphate (dHAPTP) has been synthesized from 6-chloropurine by a procedure involving both enzymatic and chemical reagents. In a series of experiments involving several different DNA polymerases including 3 procaryotic and 2 eucaryotic enzymes, it was shown that dHAPTP is ambiguous in its base-pairing characteristics, since it can replace both dATP and dGTP in DNA synthesis. It was also shown that different enzymes have different capacities to distinguish dHAPTP from the canonical deoxynucleoside triphosphates. These results are consistent with (but do not prove) the hypothesis that the mechanism of 6-N-hydroxylaminopurine mutagenesis seen in both eucaryotic and procaryotic organisms is due to its conversion, in vivo, to a deoxynucleoside triphosphate which is incorporated ambiguously for dATP and dGTP during DNA replication.     

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.     

Transient expression of Drosophila melanogaster deoxynucleoside kinase gene enhances cytotoxicity of nucleoside analogs

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-beta-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-beta-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. V. Synthesis and absolute configuration of Rp and Sp diastereomers of an ethyl phosphotriester (Et) modified EcoRI recognition sequence, d[GGAA(Et)TTCC]. A synthetic approach to regio- and stereospecific ethylation-interference studies.

Protected deoxynucleoside 3'-O-ethyl-N,N-diisopropylphosphoramidite reagents were prepared for use in the automated synthesis of ethyl phosphotriester (Et) modified oligonucleotides. The title diastereomers were separated by reversed-phase HPLC, and chirality at phosphorus was assigned by an improved configurational correlation scheme that was verified by NMR spectroscopic studies (accompanying paper, Part VI). This generally applicable correlation scheme involved enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; phosphite triester sulfurization to obtain diastereomeric O-ethyl phosphorothioates, d[AS(Et)T], which were separated by HPLC for stereoretentive oxidation with H2O2 to give d[A(Et)T], and stereoretentive de-ethylation with PhSH-Et3N to give diastereomeric phosphorothioates, d[AST], whose configurations at phosphorus had been assigned previously. Neither the Rp-Rp nor Sp-Sp duplex, (d[GGAA(Et)TTCC])2, was cleaved by EcoRI endonuclease under conditions that led to cleavage of both the unmodified duplex, [d(GGAATTCC)]2, and the mixture of diastereomeric phosphorothioate-modified duplexes, [d(GGAASTTCC)]2. Cleavage of the latter substrates was Sp-selective.     

Synthesis and separation of diastereomers of deoxynucleoside 5'-O-(1-thio)triphosphates.

Treatment of unprotected nucleosides with an excess of phosphorous acid and stoichiometric proportions of N,N'-di-p-tolylcarbodiimide in anhydrous pyridine gives predominantly deoxynucleoside monophosphites and minor amounts of 5' :3'-diphosphites; for deoxyadenosine and deoxyguanosine, the monophosphite products are exclusively 5'-phosphites, whereas for deoxycytidine and thymidine, the yields of the 5'-phosphites are 85% and 92% respectively. Sulfurization of these deoxynucleoside monophosphites with sulfur in the presence of trialkylamines and trimethylsilyl chloride in dry pyridine nearly quantitatively produces deoxynucleoside phosphorothioates. Condensation of these phosphorothioates with pyrophosphate forms diastereomers of the alpha-thio-derivatives of deoxynucleoside triphosphate. The individual diastereomers of each deoxynucleoside 5'-O-(1-thio)triphosphate can be separated, on a preparative scale, by ion exchange chromatography.     

Analysis of the complex transcription termination region of the Escherichia coli rrnB gene.

The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector. The in vivo terminating efficiency was assayed by measuring the galactokinase activity encoded by the downstream galK gene. Termination efficiencies of all fragments were compared in two constructs, differing in the presence or absence of readthrough translation over the investigated terminator signal. The following main conclusions were drawn. (a) T1 and T2 are both efficient terminators in isolated forms. (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation. Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated. (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced. (d) Translation radically decreases the efficiency of the terminators. (e) Several sequences in the rrnB gene, upstream of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation.     

Accuracy of DNA polymerase-alpha in copying natural DNA

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.     

The preparation of 5-cyanouracil and 5-cyano-2''-deoxyuridine from the corresponding 5-iodo derivative and cuprous cyanide.

5-Cyanouracil has been prepared in high yield from cuprous cyanide and 5-iodouracil. The deoxynucleoside has been similarly prepared form 5-iodo-2'-deoxyuridine and this has enabled these compounds to be labelled with (14-C) cyanide. Attempts have been made to incorporate 5-cyanouracil into Escherichia coli 15T and into Mycoplasma mycoides var. capri DNA under conditions in which several other 5-substituted uracils have been incorporated, but without success. Similarly 5-cyano-2'-deoxyuridine could not be incorporated into the DNA of T3 phage under conditions in which 5-bromo-2'-deoxyuridine is easily incorporated. These results suggest that the criteria for a 5-substituted uracil to be incorporated into DNA in vivo depends on some factor other than the size of the substituent.     

Specific inhibition of DNA biosynthesis induced by 3'-amino-2',3'-dideoxycytidine

3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation.     

The mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha.

The mode of action by aphidicolin on DNA polymerase alpha from the nuclear fraction of sea-urchin blastulae was studied. The inhibition of DNA polymerase alpha by aphidicolin was uncompetive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase alpha by competing with dCTP with a Ki value of 0.5 microgram/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC). (dG)12-18 or poly(dA) . (dT)12-18. Similar results were obtained for DNA polymerase alpha (B form) from mouse myeloma MOPC 104E.     

Studies on the Pathway of Incorporation of 2-Aminopurine into the Deoxyribonucleic Acid of Escherichia coli

A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.     

Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.     

Transient Expression of Drosophila melanogaster Deoxynucleoside Kinase Gene Enhances Cytotoxicity of Nucleoside Analogs

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-β-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-β-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.     

Control of Early Gene Expression of Bacteriophage T4: Involvement of the Host rho Factor and the mot Gene of the Bacteriophage

Many early mRNA species of bacteriophage T4 are not synthesized after infection of Escherichia coli in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA beta-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA beta-glucosyltransferase could be formed in the presence of chloramphenicol if the E. coli host cell carried a mutation in the structural gene for the RNA chain termination factor rho. This was interpreted to mean that T4 protein(s) with anti-rho activity is normally required for the expression of these two early genes. An altered rho-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the mot gene of T4 seemed to be involved, since the primary infection of E. coli cells with the mot gene mutant tsG1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated mot gene.