Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl.

The synthesis of protected nucleoside phosphoramidites bearing various markers such as biotinyl, dinitrophenyl, dansyl and pyrenyl groups are reported. These labelled deoxynucleosides phosphoramidites were used for solid phase oligonucleotide synthesis in the same way than the usual protected phosphoramidities without any change in the synthetic cycle and the deprotection step. The new labelled building blocks described herein have been used in conjunction with the labile base protected phosphoramidites ('PAC phosphoramidites') which allowed mild ammonia deprotection, especially recommended for the dinitrophenyl-labelled oligonucleotides. Multiple labelling (i.e. 10 to 20 biotins) can be efficiently and easily performed, on the same oligonucleotide which results in an increase of sensitivity. The polylabelled oligonucleotides are chemically well defined and gave increased signal and low background coloration for in situ hybridisation. The modified oligonucleotides can still be kinased in the normal way as the reporter groups are on the heterocycles.     

Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis.

A new activator for the coupling of phosphoramidites to the 5'-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4, 5-dicyanoimidazole (DCI) as the activator, compared with 1 H -tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of oligonucleotides. The addition of 0.1 M N -methylimidazole to 0.45 M 1 H -tetrazole also results in higher product yields during oligonucleotide synthesis than observed with 1 H -tetrazole alone.     

Synthesis and Antibody Mediated Detection of 2,4-Dinitrophenyl (DNP) Labelled Oligonucleotides

Abstract

Different DNP phosphoramidites based on non-nucleoside and nucleoside backbone molecules are developed and used in the multiple labelling of oligonucleotides during the solid phase synthesis. It is demonstrated that the antibody mediated detection of DNP labelled oligonucleotides is comparable to that of digoxigenin, biotin and fluorescein.     

Reagents for the selective immobilization of oligonucleotides on solid supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5'-terminus and an anchor group at the 3'-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.     

Reagents For The Selective Immobilization Of Oligonucleotides On Solid Supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5′-terminus and an anchor group at the 3′-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.     

The production of PCR products with 5' single-stranded tails using primers that incorporate novel phosphoramidite intermediates.

We have prepared several novel phosphoramidites and have synthesised oligonucleotides incorporating them internally. The presence of these residues in an oligonucleotide template presents an impossible barrier to primed synthesis by Taq DNA polymerase. When extended as polymerase chain reaction products, these oligonucleotides no longer serve as templates for the polymerase beyond the insertion sites of the modified intermediates, thereby producing single-stranded tails on amplification products. These tails can then be used for solid phase capture and colorimetric detection of PCR products.     

Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis.

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon. Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis. We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons. In six of these eight oligonucleotides, we reduced to varying extents the concentrations of those nucleotides in the target positions that would form base pairs with the template. From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs. Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.     

DNA containing side chains with terminal triple bonds: Base-pair stability and functionalization of alkynylated pyrimidines and 7-deazapurines

The synthesis of a series of oligonucleotides containing 5-substituted pyrimidines as well as 7-substituted 7-deazapurines bearing diyne groups with terminal triple bonds is reported. The modified nucleosides were prepared from the corresponding iodo nucleosides and diynes by the Sonogashira cross-coupling reaction. They were converted into phosphoramidites and employed in solid-phase synthesis of oligonucleotides. The effect of the diyne modifications on the duplex stability was investigated. The modified nucleosides were used for further functionalization using the protocol of Huisgen-Sharpless [2+3] cycloaddition ('click chemistry').     

Observation and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection

Commercially available 'fast-deprotecting' phosphoramidites are useful for synthesizing oligonucleotides containing alkali-sensitive nucleotides. However, N-acetylated oligonucleotides were observed during solid-phase synthesis using 'fast-deprotecting' phosphoramidites in conjunction with K2CO3/MeOH ('ultra-mild') deprotection. Transamidation was localized at deoxyguanosine, which is protected as its isopropylphenoxyacetyl amide. Substitution of trimethylacetic anhydride for acetic anhydride and appropriate modification of the automated synthesis cycles eliminated this problem.     

Porphyrin substituted phosphoramidites: new building blocks for porphyrin-oligonucleotide syntheses

Thymidine phosphoramidites containing trispyridylphenyl and tetraphenylporphyrin chromophores attached via a short amide linker in the 3'-position have been synthesized and used as building blocks in solid-phase synthesis of self-complementary 8-mer oligonucleotides 3'-T-5'-GCGCGCA-3' and 5'-ACGCGCGT-3'. To our knowledge, these are the first porphyrin-oligonucleotide conjugates carrying the porphyrin chromophores in the 3'-position. Chain assembly was achieved by automated solid-phase synthesis and by inexpensive straightforward 'in flask' modification of commercially available solid supported oligonucleotides. This approach allows the synthesis of modified oligonucleotides without using costly instrumentation for automated DNA synthesis. Porphyrin-containing self-complementary oligonucleotides are expected to be a valuable model for drug binding studies and determination of conformational changes in DNA sequences using circular dichroism.     

Synthesis and properties of bile acid phosphoramidites 5'-tethered to antisense oligodeoxynucleotides against HCV.

Recently, we synthesized antisense oligonucleotides (AS-ODNs) directed against the non-coding-region (NCR) and the adjacent core region of the hepatitis C virus (HCV) RNA. Backbone modifications like phosphorothioates, methyl- and benzylphosphonates were introduced three at each end of the sequence. For improvement of liver specific drug targeting and/or hepatocellular uptake efficient AS-ODNs were covalently conjugated to biomolecules such as cholesterol or bile acids. The use of base-labile alkylphosphonates afforded mild conditions for deprotection of bile acid conjugated AS-ODNs. Here, we describe a convenient synthesis of new cholic acid and taurocholic acid phosphoramidites. Derivatization to taurocholic acid was effected directly before phosphitylation reaction, which is the last step of the phosphoramidite synthesis. These building blocks were coupled to the 5'-position of AS-ODNs in the last step of solid-phase synthesis. After mild deprotection, purification and characterization the properties of these modified AS-ODNs like their lipophilicity or their ability to form stable duplices to DNA and RNA were investigated. Enhanced lipophilicity and formation of stable duplices and heteroduplices makes bile acid conjugated AS-ODNs interesting as antiviral antisense therapeutics against HCV.     

寡核苷酸药物及寡核苷酸制备技术研究进展

寡核苷酸是生物医学和生命科学研究中调节基因表达的基本工具,并被开发为基因靶向治疗药物,用于治疗病毒、肿瘤和遗传病。寡核苷酸药物主要包括反义寡核苷酸、小干扰RNA、核酶、脱氧核酶、反基因、Cp G寡核苷酸、转录因子诱饵和核酸适配体等。天然的寡核苷酸在体内很容易被降解,特异性低,且有毒副作用。因此,药物寡核苷酸通常带有特定的修饰基团,如硫代磷酸二酯键、氟代、甲基以及锁核酸等,以增强寡核苷酸在体内的稳定性,提高特异性,并降低其毒副作用。目前,寡核苷酸主要采用化学方法合成,但化学合成的寡核苷酸初产物纯度低,而纯化十分困难。大规模核酸合成仪和纯化设备十分昂贵,因而大量合成和纯化寡核苷酸的成本高昂,大大限制了寡核苷酸药物的研究和应用。尽管已经涌现了多种多样的核酸扩增和检测方法,但用于扩增寡核苷酸的方法极少,且均不适合大量制备寡核苷酸。一种新的基于热循环的寡核苷酸扩增方法,称为"聚合酶-内切酶扩增反应"(Polymerase-endonuclease amplification reaction,PEAR),能够使寡核苷酸等小分子核酸在双酶催化下,利用独特的"滑动-切割机制"进行自我复制,并实现指数扩增。PEAR反应简单、高效、稳定。该方法已成功制备硫代和氟代修饰的寡核苷酸,与化学合成法相比,该技术不依赖于大规模DNA合成仪,降低了生产成本,适合大量生产高纯度的寡核苷酸,将有助于推动寡核苷酸药物的研究和应用。     

Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

Trinucleotide phosphoramidites representing codons for all 20 amino acids have been prepared and used in automated, solid-phase DNA synthesis. In contrast to an earlier report, we show that these substances can be used to introduce entire codons into oligonucleotides in excess of 98% yield, and are ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.     

Improved synthesis of 2'-amino-2'-deoxyguanosine and its phosphoramidite

2'-Amino-2'-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of biochemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 2'-amino-2'-deoxyuridine by transglycosylation, but with limited overall efficiency and convenience. Using simple modifications of known reaction types, we have developed useful protocols to obtain 2'-amino-2'-deoxyguanosine and two of its phosphoramidite derivatives with greater convenience, fewer steps, and higher yields than reported previously. These phosphoramidites provide effective synthons for the incorporation of 2'-amino-2'-deoxyguanosine into oligonucleotides.     

Modifications of guanine bases during oligonucleotide synthesis.

Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions. Methods for the detection of two types of guanine modifications are described. The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing. The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA. Though the chemical structures of the two modifications are not known, they may be related. Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry. The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W. (1980) Nucleic Acids Res. 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases. We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.     

Fluorescent dye phosphoramidite labelling of oligonucleotides.

A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.     

Exploiting the chemical synthesis of RNA.

A number of nucleotide phosphoramidites are now available that permit the chemical synthesis of RNA, modified RNA and RNA-DNA chimeric oligonucleotides. Since the chemical strategy allows the introduction of a particular modification at any given site in a nucleotide polymer, very subtle and specific questions regarding structure-function relationships in RNA may be addressed.     

Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage.

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.     

Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.     

Methoxyoxalamido chemistry in the synthesis of novel amino linker and spacer phosphoramidites: a robust means for stability, structural versatility, and optimal tether length

We have developed a general route to the synthesis of novel amino linker and spacer phosphoramidites utilizing methoxyoxalamido (MOX) chemistry. The synthesis makes use of readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of phosphoramidites. Among these are monomers with exceptionally long (up to 56 atoms in length) amphipathic tethering arms. The chemistry bestows exceptional control over the physical characteristics within the tethers through the selection of appropriate building blocks. Furthermore, MOX chemistry enables fairly rapid assembly of these discrete-length tethers in a modular fashion. All novel phosphoramidites were successfully used in automated syntheses of 5'-modified oligonucleotides.