Purine Oligonucleotides Research Articles

Sequence dependence of isothermal DNA amplification via EXPAR

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

Decrease of carbon nanotube UV light absorption induced by π–π-stacking interaction with nucleotide bases

UV–vis absorption spectra of hybrids formed by carbon single-walled nanotubes (SWCNTs) and single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), and synthetic homopolynucleotides (poly(rC) and poly(rG)) have been studied. In the 300–400 nm spectral range a decrease of SWCNT light absorption because of their interaction with polymers was observed. The revealed decrease of nanotube absorption is explained by SWCNT hypochromism that results from π–π-stacking interaction of polymer nitrogen bases with the tube surface. Molecular dynamic simulation demonstrates a spontaneous wrapping of polynucleotides around the nanotube. Simulation showed that pyrimidine oligonucleotide (d(C) 25) manifests a higher rate of minimizing the hybrid structure with the wrapped polymer structure around the tube than the purine one (d(G) 25). Such polymer behavior can be explained by the purine oligonucleotide property to form a stable ordering self-stacking structure which prevents structural re-orientation, in contrast to pyrimidine polynucleotides.

The role of polyamines, Na+ and K+ in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine.purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na(+) and K(+). In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 10(9)/M range. In contrast to previous reports, K(+) did not inhibit triplex formation and at 150 mM the binding constants were again in the 10(9)/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K(+) the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K(+) was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 microM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K(+) may not be detrimental. However, it may be difficult to achieve binding constants above approximately 10(9)/M even in the presence of polycations.

Bridging the Prebiotic and RNA Worlds: Prebiotic RNA Synthesis on Clay

Progress towards the laboratory demonstration of the steps in the prebiotic origin of the RNA world is reviewed. Montmorillonite clay catalyzes the formation of RNAs containing 6–14 monomer units from the activated mononucleotides of A, C, G, I and U. The RNAs formed have 3′, 5′- and 2′, 5′-links, pyrophosphate links and have both linear and cyclic chains. The purine oligonucleotides have more 3′, 5′- links while the pyrimidine nucleotides have more 2′, 5′-linkages. Template-directed synthesis on the heterogeneous oligo(C)s formed on mont- morillonite yields the corresponding oligo(G)s. The dimer fraction formed in the reaction of a binary mixture of a purine and pyrimidine nucleotide shows sequence selectivity with about a 20 fold excess of the 5′-purine-pyrimidine dimer over that of the 5′-pyrimidine-purine dimer. RNAs as long as 50 mers are formed by the elongation of a decamer bound to montmorillonite by the daily addition of activated monomer to it over a 14-day time period.

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.

8-Aza-7-deazapurine DNA: Synthesis and Duplex Stability of Oligonucleotides Containing 7-Substituted Bases

The 7-substituted 8-aza-7-deazapurine phosphoramidites 1a – 3c as well as the phosphoramidite 4a were synthesized. In comparison to the parent purine oligonucleotide duplexes, the 7-substituted 8-aza-7-deazapurine residues lead to a significant duplex stabilization.

A new approach to overcome potassium-mediated inhibition of triplex formation.

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex : initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCI on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3'- or 5'-end of the 20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was demonstrated that a 'zipper' TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the 'zipper' oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3'-end and six nucleotides at the 5'-end. We suggest that this type of structure may prove useful for in vivo applications.

Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto-oncogene promotor.

In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro[Svinarchuk, F., Bertrand, J.-R. and Malvy, C.(1994)Nucleic Acids Res., 22, 3742-3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3' terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.

Calorimetric analysis of triple helices targeted to the d(G3A4G3).d(C3T4C3) duplex.

We present a thermodynamic analysis based on differential scanning calorimetry (DSC) of three short intermolecular DNA triplexes targeted to the same DNA duplex: d(C+3T4C+3)*d- (G3A4G3).d(C3T4C3) (PYR), d(G3A4G3)*d(G3A4G3).d(C3T4C3) (PUR), and d(G3T4G3)*d(G3A4G3).d(C3T4C3) (PUR/PYR). Enthalpies, delta H, and entropies, delta S, are measured by model-free integration of the DSC curves and are compared to the same quantities determined by van't Hoff analysis of the DSC curves and, in the case of the PYR and PUR/PYR triplexes, UV melting curves as well. In the case of the PUR triplex, which exhibits monophasic melting behavior, the calorimetric delta H and the calorimetrically determined van't Hoff delta H are in excellent agreement, indicating an all-or-none transition for this triplex. For the PYR and PUR/PYR triplexes, which melt in a biphasic manner, the calorimetrically determined van't Hoff delta H values are somewhat larger than the model-independent calorimetric delta H values. In those cases, however, good agreement is found between the calorimetric delta H values and the spectrophotometrically determined van't Hoff delta H values. The calorimetrically determined delta H values, expressed per mole of triplet, for the three triplexes are 4.5, 3.8, and 2.4 kcal/mol for the PUR, PYR, and PUR/PYR triplexes, respectively. The same order of stability is observed in terms of delta G and Tm values. The high stability of the PUR triplex at neutral pH indicates that purine oligonucleotides may be the most effective at targeting duplex regions for triple helix formation in vivo.

Interstrand complex formation of purine oligonucleotides and their nonionic analogs: the model system of d(AG)8 and its complement, d(CT)8.

We have investigated the role of purines in interstrand complex formation with regard to substitution of the negatively-charged, phosphodiester backbone by a nonionic, internucleoside linkage. Using the purine oligomer, d(AG)8, its methylphosphonate analog, d(AG)8, and the complementary pyrimidine oligomer, d(CT)8, as a model system, the stoichiometry, conformation, and stability of complexes formed at pH 8 were studied by spectroscopic and electrophoretic methods. When there is only one oligomer species in solution, d(AG)8 behaves as a single-stranded molecule. In contrast, the d(AG)8 oligomer readily forms an intermolecular self-complex, particularly in the presence of magnesium ion. Using either purine oligomer, duplexes can form with the d(CT)8 strand which differ in terms of their conformation and in the dependence of their thermal stability on sodium and magnesium ions. All studies show that a stable triplex forms with a 1:2 d(CT)8:d(AG)8 stoichiometry which does not require high concentrations of sodium or magnesium ions. Triplex formation between the d(CT)8 strand and two d(AG)8 strands was not observed. Native gel electrophoresis suggests that a 1:1:1 d(CT)8:d(AG)8:d(AG)8 complex may be formed. In regard to triplex formation, the advantage of the methylphosphonate backbone on the purine strand is clearly demonstrated.

The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure.

In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 068-14,071]. The target for the invariable part of these oligonucleotides, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here were report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the triplexes. The melting temperatures of the triplexes were found to be the same as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8 degrees C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation. Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the third strand oligonucleotide: the CD spectrum shows a 'classical' A-DNA shape with the 20-mer. This is not observed with the purine/pyrimidine stretch of the HIV-1 DNA which keeps a B-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purine(purine-pyrimidine) triplex.

A short purine oligonucleotide forms a highly stable triple helix with the promoter of the murine c-pim-1 proto-oncogene.

A homopurine-homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5'-GGG-GAGGGGGAGG-3' was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.

Preparative Isolation of Purine Oligonucleotides from Partial Hydrolyzates of Depyrimidinated DNA

Abstract DNA is chemically degraded to a mixture of pure oligonucleotides. From the resulting partial hydrolyzate, defined oligonucleotides with two to six monomer units and defined mixtures of sequence-isomeric purine oligonucleotides are isolated in preparative amounts. The partial hydrolyzate is fractionated by a chromatographic separation route which uses anion-exchange chromatography on DEAE- and QAE-Sephadex at different pH values, paper chromatography, and reversed-phase HPLC. The sequence of the isolated oligonucleotides and the composition of the mixtures of sequence-isomers were determined from chromatographic data, absorption characteristics, enzymatic degradation, and “fingerprints.”

Präparative isolierung von tetra-, penta- und hexapurin-oligonucleotiden aus partialhydrolysaten depyrimidinierter heringsspermen-DNA

Preparative isolation of tetra-, penta- und hexapurine oligonucleotides from partial hydrolysates of depyrimidinated herring sperm DNA Herring sperm DNA is chemically degraded to a complex mixture of purine necleotides. The oligonucleotides are separated from the partial hydrolysates by column chromatography. The resulting mixture of trimer to hexamer purine oligonucleotides is subsequently fractionated on QAE-Sephadex into different mixtures of sequence-isomeric purine oligonucleotides. In a final separation, which uses reversed-phase (Nucleosil C 18) high-performance liquid chromatography, these mixtures are separated under isocratic conditions into 35 pure defined purine oligonucleotides with four to six monomer units, 14 defined mixtures of sequence-isomeric purine oligonucleotides and several unidentified products. Purity and sequence of the isolated oligonucleotides are determined by the “fingerprint” method. The results of the high-performance liquid chromatographic and the “fingerprint” methods of the isolated oligonucleotides are discussed.

Isolierung von oligonucleotiden aus DNA-partialhydrolysaten mit hilfe der template-chromatographie

Isolation of oligonucleotides from partial hydrolysates of DNA using template chromatography Purine oligonucleotides are adsorbed at 0°C on poly(vinyl alcohol)-p(dC) n-DEAE-cellulose, and pyrimidine oligonucleotides on oligo(guanylic acid) gel according to the base-pairing mechanism, if their sequences contain at least three or more homologous, consecutive guanylic or cytidylic moieties. By increasing the temperature all base-paired oligonucleotides are desorbed. With this template chromatography mixtures of defined purine- or pyrimidine oligonucleotides could be isolated from fractionated partial hydrolyzates of herring sperm DNA. Afterwards these mixtures are rechromatographed on QAE-Sephadex and/or Nucleosil C 18. Using this approach oligonucleotides up to nine monomer units can be isolated either as single substances or as mixtures with defined composition on a preparative scale, which would be not possible with the already existing separation procedures when partial hydrolyzates of herring sperm DNA as starting material are used. Purity and sequence of the isolated oligonucleotides are determined by the “fingerprint” method.

Präprative isolierung von desoxiriboadenylsäuren aus hydrolysaten oxidierter heringsspermen-DNA

Preparative isolation of deoxyriboadenylic acids from hydrolysates of oxidised herring sperm DNA The preparative-scale chemical degradation of DNA from herring sperm to mixtures of purine oligonucleotides or deoxyriboadenylic acids is described. The deoxyriboadenylic acid mixture was separated into six fractions according to increasing ionic charge by column chromatography on QAE-Sephadex A-25. Of the first fraction, 75% was deoxyriboadenosine monophosphate; of the second, 99% consisted of a mixture of p(dA) 2 and (dA) 2p, while of the third 93% was pdAp. The remaining fractions contained more or less complex mixtures of longer-chain oligonucleotides which could be further separated by subsequent re-chromatography on QAE-Sephadex at pH 9.6. By application of paper chromatography to the fractions obtained from column chromatography, the pure nucleotide phosphate pdAp, p(dA) 2p, p(dA) 3p, p(dA) 4p and the mixtures of sequence isomers p(dA) 2, (dA) 2p; p(dA) 3, (dA) 3p; and p(dA) 6, (dA) 6p could be isolated preparatively. The nucleotide phosphate were converted to (dA) 2, (dA) 3 or (dA) 4 and the mixtures of sequence isomers to (dA) 2, (dA) 3 or (dA) 6 by treatment with alkaline phosphate. By this means dephosphorylated, paper chromatographically pure oligodexyriboadenylic acid may be obtained in a preparative scale from the hydrolysates. The structures of the nucleotides thus isolated were deduced from the absorbtion characteristics, the R F values and the results of enzymatic degradation.

Isolation of purine oligonucleotides after hydrazinolysis of deoxyribonucleic acid

AbstractA modified version of the classical degradation of deoxyribonucleic acid by hydrazine has been used to obtain undamaged purine‐deoxyribo oligonucleotides. This method exploits hydraziniumchloride as a reagent capable of β‐elimination of the phosphate ester group from C(3) of a deoxyribose originally carrying a pyrimidine. Alkaline hydrolysis completes the required scission of the sugar‐phosphate backbone and removes remnants of the pyrimidine skeleton still attached to some deoxyribose residues. A novel type of reaction of alkaline phosphatase from E. coli was used with advantage to free the purine‐isostychs from substitution products of deoxyribose originally carrying a pyrimidine. The resulting purine‐isostychs have been separated and characterized.

Studies on the nucleotide arrangement in deoxyribonucleic acids: XIII. Frequency and composition of purine isostichs in calf-thymus deoxyribonucleic acid

Several preparations of the apyrimidinic acid of calf-thymus DNA were subjected to hydrolysis by alkali under a variety of conditions. The purine isostichs of the general structure d(Punpn+1), and of length 1 to 8 were separated by chromatography on DEAE-cellulose and their relative frequencies determined. The results were compared with those obtained with the pyrimidine isostichs released similarly from apurinic acid. Although apyrimidinic acid poses several special problems, the results obtained with the two partial degradation products of DNA were in satisfactory agreement, especially when balances based on ultraviolet-light absorbance were compared.The various purine oligonucleotides comprising isostichs 1 to 5 were separated into groups of sequence isomers having the same total composition, and their relative frequencies were estimated and compared with the corresponding pyrimidine fractions. The results are in agreement with the suggestion that the base-pairing complementarities characteristic of the intact DNA also apply to those individual purine and pyrimidine sequences that can be studied. The findings also support the previous demonstration that the two DNA strands of calf-thymus DNA are antiparallel.

Nucleotide clusters in deoxyribonucleic acids. I. Isolation of purine oligonucleotides.

The distribution of uninterrupted purine sequences in DNA has been studied by degradation of DNA with hydrazine and KOH, followed by DEAE-cellulose chromatography with a salt gradient. Conditions of hydrolysis previously reported have not yielded reproducible results. Reasons for this lack of reproducibility have been found to include excessively severe conditions of hydrazinolysis and the failure of alkaline hydrolysis to produce discrete oligonucleotides of a single type: Punpn+1. There is a steady release of inorganic phosphorus until stable oligonucleotides of general formula Punpn−1 are produced. The modified procedure described here has given reproducible results in our laboratory. An explanation of the mechanism of degradation of DNA by hydrazine and KOH is presented.