Synthetic Polydeoxynucleotides Research Articles

Development of a microcapillary column for detecting targeted messenger RNA molecules

A capillary column in a rapid-flow system has been developed for detecting targeted messenger RNA (mRNA) molecules. The column has a structure made of two beds—one bed of porous microbeads and one bed of microbeads with a polythymidine base sequence. The targeted eukaryotic mRNA molecules are detected by two-step hybridization (sandwich hybridization) composed of polyadenosine selection of mRNA molecules and formation of a probe–target (targeted mRNA) hybrid. The sandwich hybridization, which is accomplished within 1 h, was tested using synthetic polydeoxynucleotides. Ten picomoles of the targeted polydeoxynucleotide were detected.

Interaction of cyanine dyes with nucleic acids: XXVI. Intercalation of the trimethine cyanine dye Cyan 2 into double-stranded DNA: study by spectral luminescence methods

The interaction between double-stranded (ds) DNA and the cyanine dye Cyan 2 has been studied with spectral luminescence methods. Binding constant values have been determined by fluorescence titration and dye distribution in the two-phase system ethyl acetate–water (3.6×10 4 and 1.5×10 4 M −1, respectively). Cyan 2 exhibits a small specificity for guanine-cytosine (GC) sequences in total DNA and synthetic polydeoxynucleotides poly(dA/dT) and poly(dGdC/dGdC). The DNA complexes with Cyan 2 are stable at high-ionic strength solution when NaCl is added. The dye molecule complexed with DNA is apparently shielded from the anionic quencher—iodide ion. The negative linear dichroism of the visible absorption band of aligned Cyan 2–DNA complexes indicates that the bound dye lies almost perpendicularly to the DNA helix axis. The linear dichroism of the absorption band at 260 nm suggests a considerable change in the DNA B-form. The results are consistent with an intercalative binding interaction between Cyan 2 and ds DNA.

Chemical cross-linking of ethidium to DNA by glyoxal

Ethidium was found to be efficiently cross-linked to DNA by glyoxal. Kinetic studies showed that the rate of the cross-linking reaction is strongly dependent on the glyoxal concentration. Comparative studies using a series of phenanthridines and acridines showed that NH 2 groups at both the 2 and 7 positions on the phenanthridine ring are necessary for efficient cross-linking. Studies using synthetic polydeoxynucleotides showed that the 2-amino group of guanine is absolutely required for cross-linking. Fluorescence contact energy transfer and relative viscosity experiments showed that the cross-linked drug remains intercalated into DNA. DNA gel electrophoresis and melting studies demonstrated that cross-linked ethidium does not dissociate the DNA double helix to single strands.

Poly[d(A · T)]and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(A n T) · d(AT n )], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A · T)] appears in the polymer family for n ≥ 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases ( n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A · T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A · T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.

Superoxide dismutase activity and novel reactions with hydrogen peroxide of histidine-containing nickel(II)-oligopeptide complexes and nickel(II)-induced structural changes in synthetic DNA

At physiologic pH values, histidine-containing nickel(II) oligopeptides reduced the flux of superoxide anion (O2-) generated in the hypoxanthine/xanthine oxidase system. The postulated involvement of the Ni(III)/Ni(II) redox couple in this apparent dismutation receives indirect support from electron-spin resonance data. These complexes also catalyzed the disproportionation of hydrogen peroxide, a process which generates active intermediates capable of hydroxylating p-nitrophenol and oxidizing uric acid to allantoin. An oxene moiety, namely [Nio]2+, is postulated as the active species in these H2O2-dependent reactions. Spectral analysis showed that monovalent, divalent and trivalent ions induced cooperative conformational changes in synthetic polydeoxynucleotides. For the nickel(II) ion, resistance to DNase-I activity clearly showed that an alternating G-C sequence is required for the observed transitions. It is concluded that the ability of nickel(II) peptide complexes to participate in active oxygen biochemistry suggests a possible role for nickel as a chemical promoter of cancer, whereas the capacity of the nickel(II) ion to induce conformational changes in DNA could, in principle, affect gene expression. Of course, the validity of both hypotheses require that the observed reactions be verified as biologically significant.

Competitive nucleosome reconstitution of polydeoxynucleotides containing oligoguanosine tracts

The ability of synthetic polydeoxynucleotides composed of oligoguanosine tracts of increasing length to form nucleosomes has been determined by several reconstitution procedures. When the presence of nucleosomes is determined by resistance to nuclease digestion, a protected band of approximately 150 base-pairs is detected only with difficulty for polymers containing long tracts of contiguous guanosines. However, when assayed by a shift in the electrophoretic mobility of radiolabeled polymers exchanged at 0.7 m-NaCl with authentic nucleosomes, all polymers tested are seen to form nucleosomes. Quantitative competitive reconstitution shows that the length of the tracts per se does not adversely affect their propensity to form nucleosomes, since even 150 base-pair poly(dG) · poly(dC) forms nucleosomes as well as heterogeneous-sequence DNA. However, the ability to form nucleosomes does depend on the length of the polymer repeating unit.

CD studies of double-stranded polydeoxynucleotides composed of repeating units of contiguous homopurine residues.

AbstractDouble‐stranded synthetic polydeoxynucleotides of the general form poly[d(GnCn)] · poly[d(GnCn)], poly[d(GnC)] · poly[d(GCn)], and poly[d(AnTn)] · poly[d(AnTn)] have been synthesized. When n = 4 or larger, the CD spectra of polymers of the form poly[d(GnCn)] · poly[d(GnCn)] or poly[d(GnC)] · poly[d(GCn)] closely resemble the spectrum of poly[dG] · poly[dC], suggesting that a string of four continguous guanosine residues is sufficient to induce a conformation resembling that of the polypurine · polypyrimidine. With polymers of the form poly[d(AnTn)] · poly[d(AnTn)], however, the CD spectrum only gradually approaches that of poly[dA] · poly[dT].

Template-selective stimulation of diverse DNA polymerases by the murine DNA-binding protein factor D.

Factor D, a template-selective DNA polymerase-alpha stimulatory protein from mouse liver (Fry, M., Lapidot, J., and Weisman-Shomer, P. (1985) Biochemistry 24, 7549-7556) is shown here to enhance the activities of diverse DNA polymerases with a cognate template specificity. DNA synthesis catalyzed by Escherichia coli DNA polymerase I, avian myeloblastosis virus polymerase, and some mammalian alpha- and gamma-polymerases was increased by factor D. With every enhanced polymerase, factor D increased the rate of copying of only poly(dT) among various tested synthetic poly-deoxynucleotides. Of the natural DNA templates examined, rates of copying of sparsely primed denatured DNA and of singly primed circular phi X174 or M13 bacteriophage DNA, but not of activated DNA, were enhanced. Michaelis constants (Km) of affected templates with responsive polymerases were decreased by factor D, without alteration in maximum velocity (Vmax). By contrast, factor D increased Vmax of deoxyribonucleoside 5'-monophosphate incorporation without changing Km of deoxyribonucleoside 5'-triphosphate substrates. Binding of factor D to poly(dT), poly(dA).poly(dT), and DNA, but less to poly(dA), was indicated by specific retention of their complexes on a DEAE-cellulose column. That factor D does not bind to DNA polymerase-alpha or to its complex with the DNA template was demonstrated by the failure of the factor to be coprecipitated with alpha-polymerase by anti-polymerase-alpha monoclonal antibodies in either the absence or presence of various templates. Lack of binding of factor D to the polymerase molecule was also indicated by simultaneous maximum stimulation of two competing polymerases by a limiting amount of factor. These combined results suggest that the enhancement of DNA synthesis is exerted through interaction of factor D with the template. It is proposed that this association leads to a tighter binding of the polymerase to the template and facilitates DNA synthesis.

DNA damage during aging of mouse myocardium

It is not known if DNA lesions, such as covalently modified nucleotides, change qualitatively or quantitatively during aging of post-mitotic cells. If genetic damage accumulates faster than cellular systems can repair it, a cell will eventually become defective in maintaining homeostatis. This situation would be particularly serious for cells that do not divide after they have differentiated to their terminal forms, for example, heart muscle cells. To examine this possibility a 32P-postlabeling technique was used to measure the relative level of modified nucleotides in mouse myocardial DNA as a function of age. The postlabeling analysis indicated that a modified nucleotide changed in an age-dependent manner. A nucleotide with similar chromatographic properties was induced by N-methyl-N-nitrosourea (MNU) alkylation of synthetic polydeoxynucleotides containing the base guanine but not by alkylation of synthetic DNAs lacking this base. This modified nucleotide was found to increase about 9-fold in heart DNA between 2 months and 39 months. These results suggest that the steady-state level of this type of genomic damage is greatly elevated in senescent mouse heart tissue.

Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides

A DNA methyltransferase partly purified from human placenta has been tested on a variety of synthetic polydeoxynucleotides. The results showed that: (i) the enzyme is most active as a ‘maintenance’ or ‘hemi-’ methylase but also has some de novo methylating activity; (ii) the presence or absence of A or T in the substrate strand has little influence on maintenance or de novo activity, while polymers containing C but not G in the same strand are poor de novo substrates and bind poorly to the enzyme; (iii) single-stranded polymers are about as good substrates as double-stranded ones, and the effects of nucleotide composition (particularly G and mC content) on enzyme activity with single strands are similar to those with double-stranded polymers; (iv) strands in which all the cytosines are methylated bind the enzyme well. A mechanism is suggested involving two different sites on the enzyme that recognize CG and mCG, and which rationalizes the activity of eukaryotic DNA methyltransferases towards single-stranded DNA.

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex.

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.

Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication.

The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than T4 and T5 DNA polymerases. At the same time, there was a higher turnover of dCTP than of dTTP by the E. coli enzyme. However, in all cases, the turnover of deoxynucleotides during replication of m6Gua-containing templates was more than that observed with templates without the alkylated base.

Interaction of diamidino-2-phenylindole (DAPI) with natural and synthetic nucleic acids.

The interaction of DAPI with natural and synthetic polydeoxynucleotides of different base content and sequences was studied with circular dichroism, ultracentrifugation, viscosity and calorimetry. All the polymers show two types of binding. The strength of the interaction and its resistance to ionic strength are related to the content of AT clusters in the chain. On the other hand, sedimentation measurements rule out an intercalation mechanism. A model of DAPI interaction with DNA, similar to that displayed by distamycin and netropsin, is proposed.

Convenient continuous sonication method for the preparation of medium-sized polydeoxynucleotides

We recently reported results on polydeoxynucleotides prepared for 31p NMR studies by sonication [1]. For these studies we required samples of medium length, from ca. 50-200 base pairs (bp), comparable to the size of DNA in nucleosome core particles. To obtain this size range we utilized the continuous low-temperature sonication procedure described for calf-thymus DNA by Davis and Phillips [2]. We have extended this procedure to small volumes of synthetic polydeoxynucleotides in a convenient preparative method. The apparatus used is shown in Fig. 1. The temperature was continuously monitored and kept between 0-4°C using a circulating cooling bath. Volumes down to 1 ml are possible in this apparatus, although we generally used 2-4 ml containing 5-100 A260n m units in 0.1-1.0M NaCI solution. A tapered microprobe was used with a He~t Systems-Ultrasonics W225R sonicator. The power level was optimized just below observable cavitation-bubbling (ca. 25 W). Experiments comparing the efficiency of breakage using the pulsed capability for comparable time periods (e.g. 50% duty cycle for twice the total time) indicated that continuous sonication was more efficient. The size distribution of the samples after sonication was determined by gel electrophoresis. Following development of the gel in ethidium bromide, the photographic negatives were scanned with a microdensitometer (Joyce-Loebl double beam, model IIIB) and the scans were digitized. The distances migrated were converted to base pairs relative to known standards using the formula described by Kovacic and Van Holde [3]. Results for sonication for different time periods showed that it was possible to obtain fairly well defined distributions with several base sequences [1]. For example, 10 min sonication of poly(dGdC), poly(dGdC) gave a maximum at

Changes in the helical repeat of poly(dG-m5dC) . poly(dG-m5dC) and poly(dG-dC) . poly(dG-dC) associated with the B-Z transition.

Random sequence DNA adsorbed to surfaces such as calcium phosphate has recently been shown to be cut preferentially by nucleases1 at sites separated by 10.6±0.1 nucleotides2, corresponding to the helical periodicity of the DNA. We have now applied a variation of this technique to determine the periodicity of the alternating synthetic polydeoxynucleotides poly(dG-dC) . poly(dG-dC) and poly(dG-m5dC) · poly(dG-m5dC) · We have also obtained X-ray diffraction patterns from fibres of both polymers to compare then helical parameters. Evidence from a variety of physical measurements3–5 suggests that in appropriate solvent conditions these polymers can undergo a transition from the usual B form to a new structure tentatively identified with the left-handed Z form. We find by X-ray fibre diffraction that the methylated polymer can adopt a Z form indistinguishable from that of the unmethylated polymer. We further find that when adsorbed to calcium phosphate or calcium oxalate, the B form of each polymer has a repeat of ∼10.5 base pairs (bp), while the repeat of the Z form is ∼13.6 bp.

Kinetics of dissociation of quinoxaline antibiotics from DNA

The kinetics of detergent-induced dissociation of triostins A and C and quinomycin C from DNA have been investigated. All three antibiotics dissociate from poly(dA-dT) and poly(dG-dC) in a simple first-order fashion whereas their dissociation from a natural DNA (calf thymus) is complex, requiring three exponential terms for its complete description. This behaviour is attributed to sequence-selectivity on the part of the drugs and seems to represent dissociation from different classes of intercalative binding site. The time constants of dissociation are better resolved for quinomycins than for triostins, consistent with the view that quinomycins are more sequence-specific in their interaction with DNA, but it is not possible to identify any class of binding site with the alternating purine-pyrimidine sequences of the synthetic polydeoxynucleotides. In general, the triostins dissociate an order of magnitude faster than the corresponding quinomycins. This is attributable to a larger entropy of activation, presumably reflecting greater flexibility of the octapeptide ring when the cross-bridge is a disulphide as opposed to the slightly shorter thioacetal found in quinomycins. The longest time constant in the dissociation of each of the four quinoxaline antibiotics from calf thymus DNA correlates well with its antibacterial potency, in agreement with the conclusion that the biological effects result from impairment of the role of DNA as a template for polymerase activity.

The Interaction of Daunomycin with Polydeoxynucleotides

The ability of daunomycin to bind to various DNA polymers has been sutided by thermal denaturation, spectrophotometric analysis and inhibition of the polymerisation reactions catalysed by Escherichia coli DNA polymerase I and rat liver DNA polymerase alpha. The quantitative binding measurements revealed that the antibiotic binds tightly to all synthetic polydeoxynucleotides studied. The results demonstrated that daunomycin can bind with equal affinity to dG . dC or dA . dT basepaired sequences. However, the number of binding sites per nucleotide for poly(dA) . poly(dT) is significantly lower than that found for poly(dA-dT) . poly(dA-dT), thus indicating an appreciable preference of the drug for the alternating copolymer. The inactivation of the template properties of the synthetic DNA polymers in the DNA polymerase system is consistent with their daunomycin binding ability. However, a lack of correlation was observed between the drug binding ability of different DNA polymers and the binding-induced stabilisation of the double helix to heat denaturation.

Interaction of 4′-6-diamidino-2-phenylindole to nucleic acids, and its implication to their template activity in RNA-polymerase reaction of E. coli bacteria and of friend-virus infected mouse spleen

Summary The interaction between 4′-6-diamidino-2-phenylindole-dihydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The data presented here suggest that DAPI interacts preferentially to dAT-rich sites of DNA. Interestingly, the drug exhibits a very high affinity towards the hybrid polymer, poly (rA) · poly (dT). The preferential binding of DAPI to dAT-rich sites of DNA was also observed in our enzymatic studies. The inhibitory effect of DAPI on the RNA-polymerase reaction of bacterial cells was strictly dependent on the base composition of the template used. This inhibition is of competitive type, as evidenced by the Lineweaver-Burk plots of the kinetic data. Studies with RNA-polymerases I and II of the nuclei from FLV-infected mouse spleen revealed that the inhibitory effect of DAPI in both the systems was similar. The drug : DNA ratio ( r ) required to inhibit 50% of the enzyme activity was identical for both the systems.

Influence of 4'-6'-diamidino-2-phenylindole on the secondary structure and template activities of DNA and polydeoxynucleotides.

The interaction between 4'-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI:DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380-392 nm for DAPI-DNA COMplexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex. Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.