Support-bound Oligonucleotide Research Articles

Convenient Solid-Phase Attachment of Small-Molecule Ligands to Oligonucleotides via a Biodegradable Acid-Labile P-N-Bond

One of the key problems in the design of therapeutic and diagnostic oligonucleotides is the attachment of small-molecule ligands for targeted deliveries in such a manner that provides the controlled release of the oligonucleotide at a certain moment. Here, we propose a novel, convenient approach for attaching ligands to the 5'-end of the oligonucleotide via biodegradable, acid-labile phosphoramide linkage. The method includes the activation of the 5'-terminal phosphate of the fully protected, support-bound oligonucleotide, followed by interaction with a ligand bearing the primary amino group. This technique is simple to perform, allows for forcing the reaction to completion by adding excess soluble reactant, eliminates the problem of the limited solubility of reagents, and affords the possibility of using different solvents, including water/organic media. We demonstrated the advantages of this approach by synthesizing and characterizing a wide variety of oligonucleotide 5'-conjugates with different ligands, such as cholesterol, aliphatic oleylamine, and p-anisic acid. The developed method suits different types of oligonucleotides (deoxyribo-, 2'-O-methylribo-, ribo-, and others).

A Versatile Solid-Phase Approach to the Synthesis of Oligonucleotide Conjugates with Biodegradable Hydrazone Linker.

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5′-end. We demonstrated the proof-of-principle of this approach by synthesizing 5′-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2′-O-methylribo-, and others) with ligands of different nature.

A convenient solid phase approach to obtain lipophilic 5′-phosphoramidate derivatives of DNA and RNA oligonucleotides

ABSTRACTThis paper explores the potential of a modified phosphotriester approach to the synthesis of 5′-phosphoramidate derivatives of DNA and RNA oligonucleotides. The modification of 5′-deprotected support-bound oligonucleotides is done in two steps: i) conversion of the 5′-OH group of an oligonucleotide into an activated phosphodiester, and ii) treatment of the activated phosphodiester with an aminocompound. The approach is efficient and compatible with conventional solid phase oligonucleotide synthesis. It can be used for the conjugation of therapeutically relevant oligonucleotides with functional moieties or carrier constructions, which are to be removed after endocytosis.

Solid-Supported 2′-O-Glycoconjugation of Oligonucleotides by Azidation and Click Reactions

2'-O-[(2-Bromoethoxy)methyl]cytidine and 2'-O-[(2-azidoethoxy)methyl]cytidine have been prepared and introduced as appropriately protected 3'-phosphoramidite (1) and 3'-(H-phosphonate) (2) building blocks, respectively, into 2'-O-methyl oligoribonucleotides. The support-bound oligonucleotides were subjected to two consecutive conjugations with alkynyl-functionalized monosaccharides. The first saccharide was introduced by a Cu(I) promoted click reaction with 2 and the second by azidation of the 2-bromoethoxy group of 1 followed by the click reaction. The influence of the 2'-glycoconjugations on hybridization with DNA and 2'-O-methyl RNA targets was studied. Two saccharide units within a 15-mer oligonucleotide had a barely noticeable effect on the duplex stability, while introduction of a third one moderately decreased the melting temperature.

Synthesis of Oligonucleotide Glycoconjugates Using Sequential Click and Oximation Ligations

Oligodeoxyribonucleotide glycoconjugates bearing two trivalent glycoclusters have been synthesized by two alternative methods based on solid-supported oximation of aminooxy functionalized oligonucleotides with glycoclusters constructed by click chemistry. In more detail, the trivalent glycoclusters (5 and 6) bearing three sugar pendants were first assembled by treating a 4-[tri-O-propargylpentaerythrityloxy]benzaldehyde scaffold with methyl 6-azido-6-deoxyglycopyranoside under the click reaction conditions. Two phosphoramidite reagents containing a phthaloyl protected aminooxy function, viz., 2-cyanoethyl N,N-diisopropylphosphoramidites derived from 3-[3,5-bis(phthalimidoxymethyl)phenoxy]propanol (12) and 5-(4,4'-dimethoxytrityl)-1,2-dideoxy-1-C-(2-phthalimidoxyethyl)-beta-d-erythro-pentofuranose (16), were synthesized and incorporated as branching units in appropriate places of the oligonucleotide chains. On using 12, the phthaloyl protections of the branching unit were removed and two identical glycoclusters were attached via oxime linkage to the 5'-terminus of the support-bound oligonucleotide chain. With branching unit 16, the phosphoramidite coupling and the oximation were carried out alternately, allowing introduction of two dissimilar trivalent glycoclusters close to the 3'-end of the oligonucleotide chain. The products (20, 26) were released and deprotected by ammonolysis and purified by HPLC chromatography.

A Comparison of Hybridization Efficiency between Flat Glass and Channel Glass Solid Supports

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

Tandem oligonucleotide synthesis using linker phosphoramidites

Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

RNA synthesis using 2'-O-(tert-butyldimethylsilyl) protection.

This chapter enables the reader to carry out the solid-phase synthesis of ribonucleic acid (RNA) using beta-cyanoethyl phosphoramidite chemistry combined with tert-butyldimethylsilyl protection of the ribose 2'-hydroxyl group. Phosphoramidite monomers are activated with 5-benzylmercapto-1H-tetrazole enabling fast and highly efficient coupling to the 5'-hydroxyl group of the support-bound oligonucleotide. On completion of the synthesis, the stepwise deprotection of the nucleobase, phosphate, and ribose protecting groups is carried out using optimized protocols. Subsequently the various high-pressure (performance) liquid chromatography (HPLC) procedures are described enabling the purification and analysis of the RNA. For this purpose anion-exchange and reversed-phase HPLC are used singly or in combination according to the final purity requirement of the RNA.

Overcoming Backpressure Problems during Solid-Phase Synthesis of Oligonucleotides

During solid-phase synthesis of oligonucleotides in packed-bed reactors a substantial pressure build-up is observed during acid deprotection and subsequent solvent washing as the length of the support-bound oligonucleotide increases. Maintaining high flow rates of reagents and wash solvents is crucial to achieving the necessary short acid-exposure times during these steps. Exceeding the maximum system pressure limit, especially likely to occur in the latter part of the synthesis, may lead to shutdown of the automated synthesis. Addition of inert filling materials (e.g., glass microspheres, quartz sand) to the polymeric polystyrene-based solid support reduces pressure build-up, allowing automated synthesis to go to completion without reducing flow rates or manual intervention.

Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation.

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.

A conformationally preorganized universal solid support for efficient oligonucleotide synthesis.

A novel, conformationally preorganized nonnucleosidic universal solid support for oligonucleotide synthesis was developed. The solid support featured two chemically equivalent hydroxy groups locked in syn-periplanar orientation and orthogonally protected with 4,4'-dimethoxytrityl and acetyl groups. The solid support was extensively tested in the preparation of oligonucleotides and their phosphorothioate analogues containing 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethylnucleoside residues at the 3'-terminus. Upon completion of oligonucleotide chain assembly, the support-bound oligonucleotide material was treated with concentrated ammonium hydroxide, which removed the O-acetyl protection. The deprotected hydroxy group then effected the transesterification of a phosphate linkage between the solid support and the 3'-terminal nucleoside residue to result in a facile release of the oligonucleotide to solution. The kinetics of the release process was studied in a continuous flow of concentrated aqueous ammonium hydroxide at a temperature of 300.15 K. Optimal conditions for the release of oligonucleotides depending on the chemistry of the backbone and 3'-terminal nucleoside residue were formulated.

ChemInform Abstract: Dimethoxytrityl Removal in Organic Medium: Efficient Oligonucleotide Synthesis without Chlorinated Solvents

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Aminooxy functionalized oligonucleotides: preparation, on-support derivatization, and postsynthetic attachment to polymer support.

Three novel phosphoramidites, each bearing a phthaloyl-protected aminooxy tail, were prepared and applied in automated oligonucleotide synthesis. After chain assembly, the phthaloyl protection was removed with hydrazinium acetate. Normal succinyl linker turned to be stable under these conditions, and hence the support-bound oligonucleotide could be converted to a pyrene oxime conjugates by reacting with pyrene carbaldehyde or cis-retinal. Standard ammonolytic deprotection then released the deprotected conjugate in solution. Alternatively, the crude aminooxy-tethered oligonucleotide was immobilized to microscopic polymer particles by reacting the aminooxy function with the particle-bound aldehyde or epoxide groups. These immobilized oligonuceotides were shown to serve properly as probes in a mixed phase hybridization assay.

Dimethoxytrityl Removal in Organic Medium: Efficient Oligonucleotide Synthesis Without Chlorinated Solvents

Oligonucleotides are finding widespread utility in various applications in diagnostics and molecular biology and as therapeutic agents. In standard synthesis of such oligonucleotides through phosphoramidite coupling, removal of the typical acid-labile 4,4′-dimethoxytrityl 5′-protecting group (DMTr), from the support-bound oligonucleotide plays a crucial role in each synthesis cycle in achieving high product yield and oligonucleotide quality. Although several reagents have been developed for this purpose, many have limited applicability to automated oligonucleotide synthesis on solid supports. The most commonly used reagents today are dilute solutions (2–15%) of an organic acid, typically trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5) in dichloromethane. The high volatility (boiling point 40 °C) of dichloromethane and its high toxicity and carcinogenicity pose a hazard for personnel and the environment. In addition, as oligonucleotide synthesizers are now available to allow syntheses of up to 0.5 mole scale, the quantities of chlorinated waste generated have become quite large. In this context we became interested in replacing dichloromethane as deblocking reagent solvent with a less harmful solvent while preserving product yield and quality. We now report that it is not necessary to use halogenated solvents such as dichloromethane in the deblocking step of automated oligonucleotide synthesis in order to obtain high yields of high quality oligonucleotide product.

The synthesis of peptide-oligonucleotide conjugates by a fragment coupling approach

Solid-phase synthesis of several peptide-oligonucleotide conjugates has been achieved using a peptide fragment coupling strategy on a controlled pore glass support. The conjugates contain either a hydrophobic tetrapeptide LGIG or an 8-residue basic peptide of the HIV-1 Tat protein coupled to one of two oligodeoxyribonucleotides, an oligoribonucleotide or a mixed ribo/2′- O-methyl oligonucleotide. Improved yields were obtained when internucleotide β-cyanoethyl groups were removed from the support-bound oligonucleotide prior to peptide fragment coupling, and by use of a long alkyl spacer in the linkage between peptide and oligonucleotide.

The Reactions of H-Phosphonates with Bifunctional Reagents. V. Functionalization of Support-Bound Oligonucleotides and Synthesis of Nonradioactive Hybridization Probesa

Three methods for the functionalization of oligonucleotides with aminoalkyl moieties have been developed and their efficiencies were evaluated in the preparation of non-radioactive hybridization probes. This paper is dedicated to the late Professor Tsujiaki Hata.

Improved synthesis of oligonucleoside methylphosphonate analogs

Solid-phase synthesis using pent-4-enoyl nucleoside PNT phosphonamidites allow for the facile preparation of oligonucleoside methylphosphonates, methylphosphonothioates and their chimeric analogs. With PNT phosphonamidites, nucleobase and phosphate deprotection, as well as, cleavage from the support can all be accomplished by treatment of the support-bound oligonucleotide with NH 4OH (28%, rt, 1 h).

RNA transcription from immobilized DNA templates.

We describe an RNA transcription protocol based on the multiple reuse of solid-phase synthetic DNA templates. The templates are assembled onto streptavidin-coated agarose beads via a single 5'-terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid-phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid-phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support-bound oligonucleotide with fresh coding-strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.

Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.

Hybridization properties of immobilized nucleic acids.

The 5'-end attachment of oligonucleotides to dextran supports facilitates the study of the hybridization properties of an immobilized oligonucleotide system. The hybridization properties which were studied include: hybridization capacity and kinetics, hybridization-complex stability, and reagents influencing hybridization efficiency. Results of these experiments reveal that the hybridization efficiencies of support-bound oligonucleotides were 75-80% and 40-50% for single-stranded oligonucleotide targets and long double-stranded targets, respectively. These hybridization efficiencies are dependent upon prehybridizing the support-bound oligonucleotides with dextran sulfate. In addition, comparisons of the relative hybridization efficiencies of the support-bound oligonucleotide and nitrocellulose-based systems have been made which indicate a retention of 13-28% of target sequences on the filters and a detection efficiency of 8-20%.