A modified thymine for the synthesis of site-specific thymine-guanine DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are highly cytotoxic lesions formed by a variety of important anti-tumor agents. Despite the clinical importance of ICLs, the mechanisms by which these lesions are repaired in mammalian cells have so far remained elusive. One of the obstacles in the study of ICL repair has been the limited availability of suitable methods for the synthesis of defined site-specific ICLs. We report here the synthesis of a site-specific ICL containing an ethylene-bridged G-T base pair based on the incorporation of a crosslink precursor containing a selectively reactive group on one strand using solid-phase synthesis. 3-(2-chloroethyl)thymidine was incorporated into oligonucleotides and underwent ICL formation upon annealing to a complementary strand by reacting with the base opposite to the modified T residue. A strong preference for ICL formation with a G residue opposite the reactive T was observed. Detailed characterization of the reaction product revealed that the alkylation reaction occurred with the O-6 group of G and a mechanism accounting for this preference is proposed. These G-T crosslinks introduced here will be useful for studies of ICL repair.     

Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.     

Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5′-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5′-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.     

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.     

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.     

SYNTHESIS OF SELENIUM-DERIVATIZED NUCLEOSIDES AND OLIGONUCLEOTIDES FOR X-RAY CRYSTALLOGRAPHY

We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach to introduce the selenium functionality in A, C, G, T, and U nucleosides at 5′-positions. In the incorporation of the selenium functionality, the leaving groups (bromide, mesyl, and tosyl) were readily displaced by sodium selenide, sodium diselenide, and sodium methyl selenide with yields higher than 90%. Selenium-derivatized oligonucleotides have been synthesized via phosphoramidite chemistry.     

Synthesis of selenium-derivatized nucleosides and oligonucleotides for X-ray crystallography

We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach to introduce the selenium functionality in A, C, G, T, and U nucleosides at 5'-positions. In the incorporation of the selenium functionality, the leaving groups (bromide, mesyl, and tosyl) were readily displaced by sodium selenide, sodium diselenide, and sodium methyl selenide with yields higher than 90%. Selenium-derivatized oligonucleotides have been synthesized via phosphoramidite chemistry.     

Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues.

The synthesis of the deoxynucleoside derived from the base P, 6H,8H-3,4-dihydro-pyrimido[4,5-c] [4,5-c] [1,2]oxazin-7-one, 2, and its introduction by established phosphoramidite and H-phosphonate chemistry into oligonucleotides is described. The melting transition temperatures (Tm) of a range of heptadecamer duplexes containing P/A and P/G base-pairs are compared with corresponding ones having N4-methoxycytosine (M) 1 and mismatched normal bases. P/A and P/G pairs allow closely similar duplex stabilities and have the potential to reduce the multiplicity of probes and primers based on amino acid sequences by removing the T/C degeneracy.     

Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes.

Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed using the S-triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol or 6-mercaptohexan (1) ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further derivatisation.     

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.     

Determination of oligonucleotide molecular masses by MS-MALDI

MALDI mass spectrometry (MS) of 14- to 42-mer homogeneous oligonucleotides and their mixtures was carried out using a Vision 2000 instrument (Thermo BioAnalysis, Finnigan, United States). Conditions for the determination of oligonucleotide molecular masses were optimized by applying various matrices and operation modes. The most reproducible results with minimal uncontrolled decomposition of the oligonucleotides including their apurinization during the MALDI MS registration were obtained using 2,4,6-trihydroxyacetophenone as a matrix instead of 3-hydroxypicolinic acid, usually employed in the mass spectrometry of oligonucleotides. Our approach allows the determination of molecular masses of oligonucleotides obtained by chemical synthesis and the evaluation of their component composition and purity. It was applied to the mass spectrometric analysis of oligonucleotides containing a 3'-(methyl-C-phosphonate) group or a modified 1,N6-ethenodeoxyadenosine unit.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.     

A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6-alkyldeoxyguanosine residues at preselected sites

As part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an O6-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA. Ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene G of bacteriophage phi X174 DNA. Two others are derived from plus strand sequences carrying the modification in the 12th codon of the human Ha-ras protooncogene. During this work several potentially serious side reactions, which could complicate interpretation of mutagenesis data, were observed. This paper describes a detailed study of these reactions. Since we were unable to avoid undesirable side products, we developed simple chromatographic methods for detecting and removing them.     

Solid phase-supported thymine dimers for the construction of dimer-containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA.

The ability to study the structure-activity relationships of the cis-syn thymine dimer, the major photoproduct of DNA, has been greatly aided by the availability of a building block suitable for its sequence-specific incorporation into oligonucleotides by standard automated DNA synthesis. Unfortunately, its usefulness is compromised by the fact that it takes six steps to synthesize in low overall yield and, as with all phosphoramidite building blocks, has to be used in great excess over the support in standard automated synthesis. To extend the usefulness of this building block, we have directly coupled it to standard A, C, G and T long chain alkylamine-linked controlled pore glass supports to yield a solid phase-supported dimer. We then demonstrate that 13mers containing a 3'-terminal d(T[cis-syn]TN) group synthesized with this support at 0.2 micromol scale can be efficiently incorporated into longer oligonucleotides by both primer extension with 3'-->5'exonuclease-deficient Klenow fragment or T4 polymerase and dNTPs or by enzymatic ligation with T4 DNA ligase to another oligonucleotide opposite a complementary template. The site specificity and integrity of the cis-syn thymine dimer after both primer extension and ligation was confirmed by cis-syn dimer-specific cleavage with T4 denV endonuclease V. This general approach should be applicable to the synthesis of many types of site-specific nucleic acid modifications and would be of particular use for those for which the required building blocks are expensive or difficult to make.     

Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(O6Me)GCG] and d[CGT(O6Me)GCG]

Two hexamer DNA fragments containing a carcinogenic modified base, O⁶-methyl guanine, have been synthesized by a solid-phase phosphotriester method, in which the unmodified guanine residues present were O⁶ protected with the 4-nitrophenylethyl group. These two alkylated oligonucleotides were found to have similar T_m's about 40° lower than the unmodified parent compund, d(CG)₃. Moreover, the presence of the (O⁶Me)G appears to inhibit the B→Z transition, as determined by CD spectroscopy.     

Synthesis and characterization of 8-methoxy-2'- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2'-deoxyadenosine within DNA.

The synthesis of 8-methoxy-2'-deoxyadenosine (moA) protected at N6 as an N,N-dimethylformamidine derivative and incorporation of the modified nucleoside into oligodeoxynucleotides via the phosphoramidite method are described. UV thermal denaturation studies were conducted on duplexes containing moA:G, moA:C and moA:T base pairs to determine the thermodynamic stability of duplexes containing moA relative to their adenosine (A)-containing counterparts. In the case of moA:G base pairs the effect of moA substitution is sequence dependent. In A:G mismatch-containing sequences, which have been shown by structural characterization to have a syn conformational preference at the glycosidic bond of A, moA substitution results in stabilization of the duplex. In contrast, in sequences where the A in the A:G mismatch has been shown to prefer the anti conformation moA substitution is destabilizing to the duplex. Thus moA may be a useful probe for investigating the conformational preferences of the N-glycosidic bond of adenosine within DNA. In addition, moA nucleoside is more resistant to acid-catalyzed depurination than previously described 8-bromo-2'-deoxyadenosine, allowing for facile incorporation into oligonucleotides via automated solid phase DNA synthesis.     

Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.

Oligoribonucleotides containing 3'-S-phosphorothiolate linkages are valuable probes in nucleic acid biochemistry, but their accessibility has been limited because 3'-thioribonucleoside phosphoramidites have not been available. We synthesized 3'-thioribonucleoside derivatives (C, G, and U) via glycosylations of nucleoside bases with 3-S-thiobenzoyl-5-O-toluoyl-1,2-O-diacetylfuranose 5, which was obtained from 1 ,2-O-isopropylidene-5-O-toluoyl-3-trifluoromethane-sulfonyl-alpha-D-x ylofuranose 2 by SN2 displacement with sodium thiobenzoate. Additionally, a 3'-thioinosine derivative was prepared from inosine via direct modification of the ribose, analogous to the previously reported synthesis of 3'-thioadenosine, except that the intermediate 2',3'-epoxide 9 was first protected as the 5'-O-tert-butyldiphenylsilyl ether prior to subsequent synthetic steps. This hydrophobic silyl group facilitated extraction and isolation of synthetic intermediates. After removal of the protecting groups, the 3'-thionucleosides (C, G, U, and I) were treated with 2,2'-dipyridyl disulfide to protect the free thiol group as a disulfide. The 3'-thionucleosides were converted to the corresponding phosphorothioamidites using procedures analogous to those for standard phosphoramidites. The amino groups of 3'-thiocytidine and 3'-thioguanosine were protected as benzoyl and isobutyryl amides, respectively, and the 5'- and 2'-hydroxyl groups of each nucleoside were protected as dimethoxytrityl and tert-butyldimethylsilyl ethers, respectively. The 3'-thiol group was deprotected by reduction with DTT and phosphitylated to afford analytically pure 3'-S-phosphorothioamidites 15, which were incorporated into oligoribonucleotides by solid-phase synthesis. Chemical assays and mass spectrometry of the synthetic RNA showed that ribose-3'-S-phosphorothiolate linkages were installed correctly and efficiently into RNA oligonucleotides using phosphoramidite chemistry.     

Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine]

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.