Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity

A solid-phase synthesis for a DNA analogue with a mixed guanidinium and urea backbone is reported. This material is nearly identical in structure to deoxynucleic guanidine (DNG) but the neutral urea internucleoside linkages can be used to attenuate the overall positive charge on the oligomer. The opposite charge attraction between urea containing DNG oligomers (DNGUs) and complimentary DNA can be controlled so that the affinity of DNG for DNA does not overwhelm the base-pairing discrimination necessary for specific binding. Octameric DNGU containing between 1 and 3 urea substitutions covered the range between very tight and very weak bonding. Each deletion of a positive charge reduced the thermal denaturation temperature (Tm) by approximately 5 degrees C. Mismatches in the DNA oligomers reduced the Tm values by 3 to 5 degrees C for each of the DNGU oligomers. DNGUs were found to bind in a 2:1 fashion to complimentary DNA in the same manner as DNG.     

Formation of adenosine 5'-phosphorofluoridate and the assay of adenyl cyclase in barley seeds

Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5′-phosphorofluoridate from magnesium-adenosine 5′-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3′: 5′-monophosphate was detected in barley seed homogenates or extracts.     

3'-modified oligonucleotides by reverse DNA synthesis

Reverse DNA oligonucleotide synthesis (i.e. from 5′→3′) is a strategy that has yet to be exploited fully. While utilized previously for the construction of alternating 3′-3′- and 5′-5′-linked antisense oligonucleotides, the use of nucleoside 5′-phosphoramidites has not generally been used for the elaboration of (modified) oligonucleotides. Presently, the potential of reverse oligonucleotide synthesis for the facile synthesis of 3′-modified DNAs is illustrated using a phosphoramidite derived from tyrosine. The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligonucleotide resulting from the proteinase K digestion of the vaccinia topoisomerase I–DNA covalent complex. The results confirm the nature of the structure previously assigned to this product, and establish the facility with which proteinase K is able to complete the digestion of the polypeptide backbone of the DNA oligonucleotide-linked topoisomerase I.     

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2′-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2′-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2′-deoxyinosine 9 was prepared from 2′-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-¹⁵N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.     

Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

The 3′-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3′-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3′-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5′-fragments carrying the modifications. After dephosphorylation of the 2′,3′-cyclophosphate moieties from these fragments, they are ligated to the respective 3′-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3′-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.     

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3′ and/or 5′ extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5′ position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.     

Deoxynucleic guanidine; synthesis and incorporation of purine nucleosides into positively charged DNG oligonucleotides

The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting from the corresponding 2'-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized on solid-phase using CPG as support from 3' to 5' direction using the precursor building block nucleosides.     

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2′-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2′-methylseleno cytidine phosphoramidite and improved the accessibility of the 2′-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2′-selenium derivatization had no effect on the stability of the duplexes with the 3′-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3′-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2′-Se-C and 2′-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2′-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.     

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) oligonucleotide mixed sequences

Positively charged DNG oligonucleotide mixed sequences containing A/T bases were prepared by solid-phase synthesis. Synthesis proceeds in 3'-->5' direction and involves coupling of 3'-Fmoc protected thiourea in the presence of HgCl(2)/TEA with the corresponding 5'-amine of the growing oligo chain. DNG binding characteristics with complementary DNA and with itself have been evaluated.     

The base pairing properties of 8-aza-7-deaza-2'-deoxyisoguanosine and 7-halogenated derivatives in oligonucleotide duplexes with parallel and antiparallel chain orientation

The base pairing properties of oligonucleotide duplexes containing 8-aza-7-deaza-2′-deoxyisoguanosine, its 7-bromo or its 7-iodo derivative are described. The nucleosides were synthesized on a convergent route, protected and converted into phosphoramidites. Oligonucleotides were prepared on a solid-phase and were hybridized to yield duplexes with parallel (ps) or antiparallel (aps) chain orientation. The 8-aza-7-deaza-2′-deoxyisoguanosine-containing duplexes show almost identical base pairing stability as those containing 2′-deoxyisoguanosine, while the 7-substituted derivatives induce a significant duplex stabilization both in ps and aps DNA. Self-complementary duplexes with parallel chain orientation are exceptionally stable due to the presence of 5′-overhangs. The bulky halogen substituents were found to be well accommodated in the grooves both of aps and ps DNA.     

7-Deaza-2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3′- and 5′-junctions of d(A6)·d(T6) and B-DNA

7-Deaza-2′-deoxyadenosine (1, c⁷A_d) and 3-deaza-2′-deoxyadenosine (2, c³A_d) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthsized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5′ by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c³A_d decreased the bending more than replacement by c⁷A_d. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3′-site of an d(AAAAAA)-tract whereas replacement at the 5′-site showed no significant influence [1, 2].     

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using ³²P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2′-O-methyl modifications

One of the problems that hamper the use of antisense DNAs as effective drugs is the non-specific binding of chemically-modified oligonucleotides to cellular proteins. We previously showed that the affinity of a model ssDNA-binding protein, the Ff gene 5 protein (g5p), was >300-fold higher for phosphorothioate-modified DNA (S-DNA) than for unmodified dA₃₆, consistent with the propensity of S-DNA to bind indiscriminately to proteins. The current work shows that g5p binding is also sensitive to sugar and pyrimidine modifications used in antisense oligomers. Binding affinities of g5p for 10 36mer oligomers were quantitated using solution circular dichroism measurements. The oligomers contained C-5-propyne (prC), 2′-O-methyl (2′-O-Me) or 2′-OH (RNA) groups, alone or combined with the phosphorothioate modification. In agreement with reported increases in antisense activity, the addition of prC or 2′-O-Me modifications substantially reduced the affinity of oligomers for g5p by ~2-fold compared with the same DNA oligomer sequences containing only phosphorothioate linkages. That is, such modifications moderated the propensity of the phosphorothioate group to bind tightly to the g5p. The Ff g5p could be a useful model protein for assessing non-specific binding effects of antisense oligomer modifications.     

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.     

Tandem oligonucleotide synthesis using linker phosphoramidites

Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.     

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.     

Selective Glutaraldehyde-Mediated Coupling of Proteins to the 3′-Adenine Terminus of Polymerase Chain Reaction Products

Attachment of proteins to the 3′ end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ′ modifications by solid-phase synthesis for protein attachment is limited to 45–60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3′-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N⁶ position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4°C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and β2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4°C showed no evidence of DNA–DNA or DNA–protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA–3′-protein conjugates might be easily mass-produced. The protein portion of a DNA–3′-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.