A Universal Glass Support for Oligonucleotide Synthesis

Abstract

A single type of controlled pore glass derivatized with 3-anisoyl-2′(3′)-O-benzoyluridine 5′-O-succinyl residues can be used as the support in solid phase syntheses of either oligoribo- or oligodeoxyribonucleotides.     

1,2-Diol and hydrazide phosphoramidites for solid-phase synthesis and chemoselective ligation of 2'-modified oligonucleotides

The preparation of two novel 2'-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2'-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO4 to generate 2'-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions.     

An improved CPG support for the synthesis of 3'-amine-tailed oligonucleotides.

A new controlled-pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotides bearing a 3'-aminohexyl tail. This solid support (AH-CPG) exhibits superior performance as compared to a commercially available 3'-amine CPG. The AH-CPG is prepared from 6-aminohexan-1-ol with a unique protecting group for the amine that also functions as the site of attachment to the CPG. A 3'-amine-tailed oligodeoxynucleotide (ODN) was prepared from this support using standard phosphoramidite coupling and deprotection conditions. The 3'-amine-tailed ODN was subsequently modified with an acridinylpropionic acid tetrafluorophenyl ester. Facile synthesis of the AH-CPG and the stability of the deprotected product makes this functionalized solid support especially useful for preparation of oligonucleotides bearing 3'-amine tails and other modifications.     

Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay.

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.     

Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.     

1,2-Diol and Hydrazide Phosphoramidites for Solid-Phase Synthesis and Chemoselective Ligation of 2′-Modified Oligonucleotides

Abstract

The preparation of two novel 2′-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2′-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO₄ to generate 2′-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions.     

Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA.

2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.     

Structure-based cross-linking of NF-kappaB p50 homodimer and decoy bearing a novel 2'-disulfide trapping site

Double-stranded oligodeoxyribonucleotides with engineered disulfide units were successfully used for covalent trapping of cysteine containing proteins. In particular, an efficient cross-linking of NF-kappaB p50 homodimer to a sequence-specific decoy was demonstrated. The results suggest that the synthetic oligonucleotides bearing a novel 2'-disulfide trapping site can be used as new tools to study and manipulate biological systems.     

Reactivity of oligonucleotide derivatives, containing methylphosphonate groups. VI. Increase in the effectiveness of directed action of alkylating derivatives of oligonucleotide methylphosphonate analogs on nucleic acids in the presence of effectors--3',5'-bis-N-(2-hydroxyethyl)-phenazine derivatives of oligonucleotides

Effectors for increasing the efficiency of DNA modification with the alkylating methylphosphonate analogues of oligodeoxyribonucleotides (MFAO) were suggested. Oligodeoxyribonucleotide d(pC5A8ACAATG) used as a target DNA treated with alkylating derivatives of octathymidylate having alternating methylphosphonate and phosphodiester internucleotide bonds (both Rp- and Sp-individual diastereoisomers of MFAO were used) and bearing alkylating 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide residue at the 3'-end. The reactions were carried out in the presence of an effector, hexadeoxyribonucleotide derivative PhnNH(CH2)2NHpCATTGTpNH(CH2)2NHPhn bearing two N-(2-hydroxyethyl)phenazinium (Phn) residues at the 3'- and 5'-ends and being complementary to the part of the target DNA neighbouring with octaadenylate. It was shown that Tm of the duplex formed by the target DNA, octathymidylate and effector is by 7-13 degrees C higher than in the absence of the effector, thus considerably increasing the efficiency of the intracomplex alkylation of the target (e.g., at 40 degrees C, the increase for the reagent based on the Rp-isomer is sixfold). Specificity of the target DNA modification by the MFAO alkylating derivatives in the presence of effector is same as with reagents based on oligodeoxyribonucleotides with natural internucleotide bonds.     

Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines.

A novel method for the deprotection of oligodeoxyribonucleotides has been developed. Gaseous amines such as ammonia or methylamine were employed under pressure to achieve mild and rapid deprotection conditions. For example, oligodeoxyribonucleotides having a (tert-butyl)phenoxyacetyl group for the protection of the exocyclic amino function of cytosine, adenine and guanine were released from controlled-pore glass supports and fully deprotected by ammonia or methylamine under gas phase conditions, at room temperature, within 35 or 2 min respectively.     

Rapid synthesis of oligodeoxyribonucleotides on a grafted polymer support.

Rapid synthesis of oligodeoxyribonucleotides is described by a solid phase method. Polysterene grafted on the surface of polytetrafluoroethylene has been found to be an adequate support. The properties of the support were studied in the synthesis of a number of oligonucleotides 6 to 15 nucleotides long. A flow-type semi-automatic synthesizer has been used. Each nucleotide addition involves seven steps and takes 8 hours. Oligonucleotide isolated and purified by ion-exchange chromatography amounts to 1-3 mumol per gram of support.     

Immobilization of oligodeoxyribonucleotides with multiple anchors to microchips

Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface. This report presents an oligodeoxyribonucleotide which contains a hairpin stem–loop structure with multiple phosphorothioate moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane. The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem–loop architecture with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

The isopropoxyacetic group for convenient base protection during solid-support synthesis of oligodeoxyribonucleotides and their triester analogs.

Isopropoxyacetic anhydride was successfully used for protection of exoaminofunctions of 2'-deoxyadenosine, -guanosine and -cytidine. N-isopropoxyacetylated nucleosides are stable under the conditions of the synthesis of oligodeoxyribonucleotides on the solid support. Removal of N-isopropoxyacetyl is much faster than that of commonly used benzoyl or isobutyryl groups viz. it is completed within the operation of cleavage of the oligodeoxyribonucleotide from the solid support. This observation enabled synthesis of -OCH2CH3 and -OCH2CF3 triesters, which hydrolyse partially or completely when standard deprotection conditions are applied.     

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.     

Inhibition of DNA chain elongation by a phosphotriester linkage in template oligodeoxyribonucleotides.

We synthesized two oligodeoxyribonucleotides bearing an isopropyl phosphotriester at defined positions using new phosphorobisamidite chemistry. Diastereomers were separated with HPLC and their template properties were analyzed. Priming oligodeoxyribonucleotides labeled with 32P at the 5'-end were annealed to the modified oligodeoxyribonucleotides and elongated with DNA polymerase I large fragment from Escherichia coli. Results show that the phosphotriester inhibits the DNA chain elongation partially and the extents of the inhibition are remarkably different between the two diastereomers.     

Sequence specific block of in vitro DNA synthesis with isopropyl phosphotriesters in template oligodeoxyribonucleotides.

We have synthesized four oligodeoxyribonucleotides each bearing an isopropyl phosphotriester at a defined position. These oligomers were used as templates for in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I large fragment. Results showed that the phosphotriester inhibits the DNA chain elongation and the level of the inhibition is dependent on the base 5' to the phosphotriester.     

Polymer supported DNA synthesis using hydroxybenzotriazole activated phosphotriester intermediates.

Phosphorylation of N-acyl-5'-O-DMTr-d-nucleosides, or similarly protected DNA-dimers having a free 3'-OH group, with 2-chlorophenyl-0,0-bis(1-benzotriazolyl)phosphate affords reactive 3'-phosphotriester derivatives. The latter intermediates can be used, without further purification, for the synthesis of DNA-fragments on the controlled pore glass/long chain alkylamine support. Further, 2-cyano-1,1-dimethylethoxy dichlorophosphine proved to be very suitable for the preparation of 5'-phosphorylated DNA-fragments on the same type of solid support.     

Protected deoxyribonucleoside-3'' aryl phosphodiesters as key intermediates in polynucleotide synthesis. Construction of an icosanucleotide analogous to the sequence at the ends of Rous sarcoma virus 35S RNA.

Several modifications have been incorporated into the phosphotriester strategy for chemical synthesis of oligodeoxyribonucleotides. These include high-yield methods of preparation and isolation of O5', N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates which serve as key intermediates, and the elimination of some superfluous manipulation and purification steps commonly used in the process of synthesizing oligonucleotide blocks. In addition, two new arylsulfonyl nitroimidazole derivatives have been prepared and found to be highly effective agents for internucleotide bond formation. These techniques have been applied in construction of the iconsamer d(G-C-C-A-T-T-T-T-A-C-C-A-T-T-C-A-C-C-A)-rC, equivalent to a ribonucleotide sequence located at both the 5' and 3' ends of Rous sarcoma virus 35S RNA.     

Synthesis of oligodeoxyribonucleotides bearing a functional group at 2'-position by post-synthetic modification.

A novel 2'-thio-2'-deoxyuridine derivative bearing a reactive ester at 2'-position was synthesized and incorporated into oligodeoxyribonucleotides (ODNs) for the post-synthetic modification of ODNs. This ODNs was reacted with several amines to give ODNs having a functional group at 2'-position.