Stable DNA triple helix formation using oligonucleotides containing 2'-aminoethoxy,5-propargylamino-U

We have prepared oligonucleotides containing the novel base analogue 2'-aminoethoxy,5-propargylamino-U in place of thymidine and examined their ability to form intermolecular and intramolecular triple helices by DNase I footprinting and thermal melting studies. The results were compared with those for oligonucleotides containing 5-propargylamino-dU and 2'-aminoethoxy-T. We find that the bis-substituted derivative produces a large increase in triplex stability, much greater than that produced by either of the monosubstituted analogues, which are roughly equipotent with each other. Intermolecular triplexes with 9-mer oligonucleotides containing three or four base modifications generate footprints at submicromolar concentrations even at pH 7.5, in contrast to the unmodified oligonucleotide, which failed to produce a footprint at pH 5.0, even at 30 microM. UV- and fluorescence melting studies with intramolecular triplexes confirmed that the bis-modified base produces a much greater increase in T(m) than either modification alone.     

2'',3''-Dideoxy-3'' aminonucleoside 5''-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.     

Oligonucleotides containing fluorescent 2'-deoxyisoinosine: solid-phase synthesis and duplex stability.

The fluorescent nucleoside 2'-deoxyisoinosine (2, isoId) has been incorporated into oligonucleotides. For this purpose the phosphonate 3a and the phosphoramidite 3b, as well as the polymer-linked 3d, have been synthesized and oligonucleotides were prepared by P(III) solid-phase chemistry. One or two isoId-residues were introduced into the oligomer d(T12), replacing dT either in the middle or at the 3'- and 5'-ends. The isoId-containing oligomers were hybridized with a modified d(A)12 containing the conventional nucleosides (dA, dT, dG and dC) opposite to isoId. The replacement of one dT by isoId in the centre of the duplex reduced the Tm value by approximately 15 degrees C and a decrease of approximately 25 degrees C was found when two isoId residues were incorporated. Thermodynamic data were determined from the melting curves. The destabilization was almost independent of the four naturally occurring nucleosides located opposite to isoId. The isoId (2) seems to be stacked in the duplex when dT-dA base pairs are the nearest neighbours; an internal loop is formed in the case of oligomers containing two consecutive isold residues.     

Inhibition of restriction endonuclease cleavage by triple helix formation with RNA and 2'-O-methyl RNA oligonucleotides containing 8-oxo-adenosine in place of cytidine.

The ability of homopyrimidine oligoribonucleotides (RNA) and oligo-2'-O-methyl-ribonucleotides (2'-O-methyl RNA) containing 8-oxo-adenosine (AOH) and 8-oxo-2'-O-methyl (AmOH) adenosine to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The AOH- and AmOH-substituted RNA and 2'-O-methyl RNA oligonucleotides were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. The substitutions of three cytidine residues with AOH showed higher endonuclease inhibition than the substitution of either one or two cytidine residues with AOH. In particular, the 2'-O-methyl RNA oligonucleotide with only one cytidine substituted with AmOH showed higher endonuclease inhibition than the homopyrimidine RNA and 2'-O-methyl RNA oligonucleotides and the RNA oligonucleotides containing either one or two AOH moieties. Furthermore, the AmOH-substituted 2'-O-methyl RNA oligonucleotides were stable (53%) after an incubation in 10% fetal bovine serum for 8 h, whereas the RNA oligonucleotides were completely degraded. Increased resistance to nucleases is observed with the introduction of 2'-O-methylnucleosides. This stabilization should help us to design much more efficient third strand homopyrimidine oligomer and antisense nucleic acid-based antiviral therapies, which could be used as tools in cellular biology.     

Oligonucleotides containing 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine as suitable precursors of 2'-aldehyde oligonucleotides for chemoselective ligation

2'-O-[2-(2,3-Diacetoxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite was prepared and used in solid-phase synthesis to obtain oligonucleotides containing a 1,2-diol group, which may then be converted into a 2'-aldehyde group. The oligonucleotides were conjugated efficiently to various molecules by chemoselective ligation that involves an addition-elimination reaction between the 2'-aldehyde group and a suitable nucleophile, such as a hydrazine, a O-alkylhydroxylamine or an 1,2-aminothiol. The method was applied successfully to the conjugation of peptides to oligonucleotides at the 2'-position.     

Properties of triple helix formation with oligodeoxyribonucleotides containing 8-oxo-2′-deoxyadenosine and 2′-modified nucleoside derivatives

The ability of homopyrimidine oligonucleotides containing 8-oxo-2′-deoxyadenosine (dAOH), 2′-methoxyuridine (Um). 2′-fluorouridine (Uf), 2′-methoxycytidine (Cm), and 2′-fluorocytidine (Cf) to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2′-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine residues with 8-oxo-2′-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2′-deoxyadenosine. In contrast, the oligonucleotides containing 2′-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOHUf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.

The 8-oxo-2′-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine resides with 8-oxo-2′-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2′-deoxyadenosine. By contrast, the oligonucleotides containing 2′-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOH-Uf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.     

Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine

Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives.     

Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes.     

Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine.

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.     

alpha-Oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and duplex formation.

The synthesis and hybridization properties of novel nucleic acid analogs, alpha-anomeric oligodeoxyribonucleotide N3'-->P5' phosphoramidates, are described. The alpha-3'-aminonucleoside building blocks used for oligonucleotide synthesis were synthesized from 3'-azido-3'-deoxythymidine or 3'-azido-2',3'-dideoxyuridine via acid catalyzed anomerization or transglycosylation reactions. The base-protected alpha-5'-O-DMT-3'-aminonucleosides were assembled into dimers and oligonucleotides on a solid support using the oxidative phosphorylation method.1H NMR analysis of the alpha-N3'-->P5' phosphoramidate dimer structures indicates significant differences in the sugar puckering of these compounds relative to the beta-N3'-->P5' phosphoramidates and to the alpha-phosphodiester counterparts. Additionally, the ability of the alpha-oligonucleotide N3'-->P5' phosphoramidates to form duplexes was studied using thermal denaturation experiments. Thus the N3'-->P5' phosphoramidate decamer containing only alpha-thymidine residues did not bind to poly(A) and exhibited lower duplex thermal stability with poly(dA) than that for the corresponding beta-anomeric phosphoramidate counterpart. A mixed base decamer alpha-CTTCTTCCTT formed duplexes with the RNA and DNA complementary strands only in a parallel orientation. Melting temperatures of these complexes were significantly lower, by 34-47 or 15-25 degrees C, than for the duplexes formed by the isosequential beta-phosphoramidates in antiparallel and parallel orientations respectively. In contrast, the alpha-decaadenylic N3'-->P5' phosphoramidate formed duplexes with both RNA and DNA complementary strands with a stability similar to that of the corresponding beta-anomeric phosphoramidate. Moreover, the self-complementary oligonucleotide alpha-ATATATATAT did not form an alpha:alpha homoduplex. These results demonstrate the effects of 3'-aminonucleoside anomeric configuration on sugar puckering and consequently on stability of the duplexes.     

Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.     

Application of a spectrophotometric method to the determination of the composition of oligonucleotides obtained from cysteine transfer ribonucleic acid.

1. The applications of methods for determining the composition of oligonucleotides from u.v.-absorption spectra is described. 2. In the first method absorbances at selected wave-lengths were read from the spectra of oligonucleotides in solution in 7 M-urea which had been recorded at acid and alkaline pH values. 3. In the second method absorbances were sampled automatically at regular time-intervals during scans at acid and alkaline pH of each spectrum, converted into digital signals and recorded on paper take for computer processing. The holmium spectrum in the region of the holmium peak at 333.7 nm was superimposed on each nucleotide spectrum. The position of this peak maximum was used as a standard reference point in the computer-based analysis. 4. By using either method the composition was calculated by a least-squares procedure by using a library of values for five standard nucelotides obtained in a similar manner. 5. The methods gave satisfactory compositions for mixtures of mononucleotides as well as for five dinucleoside monophosphates. 6. Methods of minimizing the effects on the nucleotide composition of spectural changes due to base stacking are discussed. 7. The compositions of some oligonucleotides obtained during an investigation of the nucleotide sequence of tRNA (Cys) were determined and agreed with the sequences found by other methods.     

Quadruplex and pentaplex self-assemblies of oligonucleotides containing short runs of 8-aza-7-deaza-2'-deoxyisoguanosine or 2'-deoxyisoguanosine.

Oligonucleotides containing 8-aza-7-deaza-2'-deoxyisoguanosine (4) were investigated regarding their self-assembly in aqueous solution. The aggregation of 4 was compared with that of oligonucleotides containing 2'-deoxyisoguanosine (2b) and 2'-deoxyguanosine (1b). For this purpose the phosphoramidite of 4 was synthesized which was protected by a dibutylaminomethylidene residue at the amino group and a diphenylcarbamoyl residue at the 2-oxo function. Solid-phase synthesis furnished oligonucleotide containing short runs of the nucleoside 4. The self-assembly of the oligonucleotide 5'-d(T(4)4(4)T2) was studied by ion-exchange chromatography. The formation of a pentaplex was observed in the presence of Cs+, while a tetraplex is formed when the counter ion is Na+ or Rb+. The cation selectivity of the oligonucleotide 5'-d(T(4)4(4)T2) was found to be different from the parent 5'-d(T(4)isoG(4)T2) which was forming the tetraplex as well as a pentaplex in aq RbCl solution.     

2'-fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity

The synthesis of oligonucleotides containing 2′-deoxy-2′-fluoro-4′-thioarabinonucleotides is described. 2′-Deoxy-2′-fluoro-5-methyl-4′-thioarabinouridine (4′S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4′S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4′S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4′-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2′-Deoxy-2′-fluoro-4′-thioarabinouridine (4′S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2′F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.     

Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.     

DNA or RNA oligonucleotide 2'-hydrazides for chemoselective click-type ligation with carbonyl compounds

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.     

Evidence of oligonucleotides containing 8-hydroxy-2'-deoxyguanosine in human urine

The product of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of <30-55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.     

Azole substituted oligonucleotides promote antiparallel triplex formation at non-homopurine duplex targets.

The ability of certain azole substituted oligodeoxy-ribonucleotides to promote antiparallel triple helix formation with duplex targets having CG or TA interruptions in the otherwise homopurine sequence was examined. 2'-Deoxyribonucleosides of the azoles, which include pyrazole, imidazole, 1,2,4-triazole and 1,2,3,4-tetrazole were synthesized using the stereo-specific sodium salt glycosylation procedure. These nucleosides were successfully incorporated using solid-support, phosphoramidite chemistry, into oligonucleotides designed to interact with the non-homopurine duplex targets. The interaction of these modified oligonucleotides with all four possible base pairs was evaluated and compared to similar data for a series of natural oligonucleotides. The oligonucleotides containing simple azoles enhanced the triplex forming ability considerably at non-homopurine targets. Binding of these modified oligonucleotides to duplex targets containing TA inversion sites was particularly noteworthy, and compare favorably to unmodified oligonucleotides for binding to duplex targets containing CG as well as TA base pairs. The selectivity exhibited by certain azoles is suggestive of base pair specific interactions. Thus, the azoles evaluated during this study show considerable promise for efforts to develop generalized triplex formation at non-homopurine duplex sequences.     

Synthesis of oligonucleotides containing 2''-deoxy-6-thioguanosine at a predetermined site.

A new approach has been devised for the synthesis of oligonucleotides containing 2'-deoxy-6-thioguanosine [d(s6G)]. The synthesis of oligonucleotides containing d(s6G) requires special protection and deprotection strategies to prevent the thione functionality from undergoing oxidation and hydrolysis. Previous attempted syntheses have neglected to address this problem. By using the cyanoethyl protecting group for the thione and phenoxyacetyl for the exocyclic amino group, it was possible to deprotect oligonucleotides with a mixture of sodium hydroxide and sodium hydrogen sulfide without any significant conversion of d(s6G) to deoxyguanosine. Application of this strategy will allow investigation of the structural as well as biological activity of d(s6G)-containing oligonucleotides.     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.