Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.     

Double-stranded oligonucleotides containing 5-aminomethyl-2'-deoxyuridine form thermostable anti-parallel triplexes with single-stranded DNA or RNA

This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions with and without MgCl(2). Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting single-stranded RNA through triplex formation.     

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.     

Branched oligonucleotides containing bicyclic nucleotides as branching points and DNA or LNA as triplex forming branch

Various Y-shaped branched oligonucleotides containing a 2'-0,3'-C-ethylene linked or 2'-0,4'-C-methylene linked bicyclic nucleotide as branching point were synthesized on an automated DNA synthesizer. Thermal denaturation experiments at 260 and 284 nm showed increased thermal stabilities of complexes formed between these Y-shaped oligonucleotides and complementary DNA compared with those formed with the corresponding linear reference. The most significant effect was observed when LNA (locked nucleic acid) monomers were used in the triplex forming branch.     

Chemical cross-linking of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.     

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).     

Interaction of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine.

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.     

Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives.

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (DeltaTm = +13 degrees C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5' end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5' end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5' end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.     

Binding properties of oligonucleotides containing a modified 2'-deoxyuridine with a thymine ended linker to pair with 2'-deoxyadenosine

Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2'-deoxyuridine harbouring at C-5 a thymine ended linker, were found to undergo base pairing with the opposite 2'-deoxyadenosine. However, the corresponding duplexes are significantly destabilised as compared to the fully natural ones.     

Nuclease stability of LNA oligonucleotides and LNA-DNA chimeras

The stabilizing properties of LNA and alpha-L-LNA oligonucleotides against endo- and 3'-exonucleases have been evaluated.     

Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides

Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time. Among the compounds prepared were a series of 2'OMe-RNA-[GAP]-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments. The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N). Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E. coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N). The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N).     

Triplex formation by oligonucleotides containing novel deoxycytidine derivatives.

Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex DNA targets with an oligodeoxyribopyrimidine containing N4-(6-amino-2-pyridinyl)deoxycytidine (1), a nucleoside designed to interact with a single C-G base pair interruption of the purine target tract, was studied by UV melting, circular dichroism spectroscopy and dimethylsulfate alkylation experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with formation of a 1-Y-Z triad, where Y-Z is a base pair in the homopurine tract of the target. Selective interaction was observed when Y-Z was C-G, although A-T and, to a lesser extent, T-A and G-C base pairs were also recognized. The circular dichroism spectra of the triplex having a 1-C-G triad were similar to those of a triplex having a C(+)-G-C triad, suggesting that the overall structures of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a triplex having the 1-C-G triad with dimethylsulfate resulted in a 50% reduction of methylation of the G residue of this triad. In contrast, the G of a similar triplex containing a U-C-G triad was not protected from methylation by dimethylsulfate. These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target duplex at the 1-C-G binding site and forms a hydrogen bond with the O6 of G. An additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.     

New synthesis of 5-carboxy-2'-deoxyuridine and its incorporation into synthetic oligonucleotides

5-Carboxy-2'-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2'-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2'-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Interaction of LNA oligonucleotides with mdr1 promoter

LNA oligonucleotides [1] can be used for targeting to double stranded DNA by the "strand invasion" mechanism. We used affinity modification by reactive oligonucleotide conjugates for investigation of oligonucleotides interaction with structured DNA. The tested LNAs and oligonucleotides of the same sequence were assayed as anti-mdr1 drugs in different cell cultures. One of the oligos, LNA79 strongly inhibited mdr1 induction in Hela cells and totally prevented activation of mdr1 in K-562.     

Novel enamine derivatives of 5,6-dihydro-2'-deoxyuridine formed in reductive amination of 5-formyl-2'-deoxyuridine

Reductive amination of 5-formyl-3',5'-di-O-acetyl-2'-deoxyuridine with primary amines and sodium triacetoxyborohydride (NaBH(OAc)(3)) afforded novel enamine derivatives of 5,6-dihydro-2'-deoxyuridine as a result of unexpected 1,4-conjugate reduction of intermediate Schiff bases in addition to the secondary amine derivatives of 2'-deoxyuridine, typical 1,2-reduction products.     

Antiviral activity of some 5-arylethynyl 2'-deoxyuridine derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by cross-linking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

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.     

Oligonucleotide duplexes containing 2'-amino-2'-deoxycytidines: thermal stability and chemical reactivity.

Thermal stabilities of oligonucleotides containing 2'-amino-2'-deoxycytidines were determined and compared to those of the unmodified oligonucleotides. The presence of the 2'-aminonucleoside destabilized duplexes in a RNA as well as a DNA context at pH 7 as well as at pH 5. The pKa of the 2'-amino group was determined by 13C-NMR spectroscopy to be 6.2. The reactivity of an oligonucleotide containing a 2'-aminonucleoside was exploited for the incorporation of rhodamine by its isothiocyanate derivative.