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.     

Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ～35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ～2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.     

DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-deaza-6-thio-2'-deoxyguanosine as gold surface anchor

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker strategy simplifies the synthesis of DNA gold nanoparticle conjugates. The phosphoramidite of 7-deaza-6-thio-2'-deoxyguanosine (6) was used in solid-phase synthesis. Incorporation of the sulfur-containing nucleosides can be performed at any position of an oligonucleotide; even multiple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow melting transition.     

Synthesis and Properties of Some (2′-5′) Linked Dinucleoside Monophosphates Modified with 3′-Difluoromethylene Groups

Abstract

The title dimers were prepared to investigate conditions required for the synthesis of 3′-difluoromethylene modified oligonucleotides on solid support. As a result a new synthetic cycle was developed that enabled the solid phase synthesis of the modified oligonucleotides.     

New light labile linker for solid phase synthesis of 2'-O-acetalester oligonucleotides and applications to siRNA prodrug development

We report on the synthesis and properties of oligonucleotides containing 2′-O-(levulinic acid) and 2′-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis of oligonucleotides containing various 2′-O-acetalesters, but can be generally adopted to the synthesis of base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with conventional linkers.     

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use.     

A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

Abstract

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogues Containing Novel Phosphotriester Internucleotide Linkage

Abstract

Enhanced cellular uptake, stable and discriminating hybridization and increased stability in biological media are of particular interest for oligonucleotides of potential therapeutic application. Additionally, toxicity or immunogenicity of the oligonucleotide analogues and their biodegradation products should be minimized by minimal alteration of the biological structure and effort and cost of bulk production should be as low as possible by using a standard automated synthesis protocol. Oligonucleotide phosphotriesters with oligoethyleneglycol substituents show promise to ideally combine all these advantages. Here we describe the hybridization properties and the stability of modified oligonucleotides containing triester internucleotide linkages substituted with α,ω-dihydroxy-(3,6-dioxa)-octan-1-yl group (“triethyleneglycol triester linkages”) towards enzymatic degradation. The triester linkages are stable towards exo- and endonucleases. Regardless of number and position of triester linkages, the modified oligonucleotides showed practically no decrease of T_m in hybridization studies with complementary biological oligonucleotides. In further enzymatic studies the modified oligonucleotides were highly stable towards nucleases in human blood serum.     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

Regulating gene expression with light-activated oligonucleotides

Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.     

Site-directed mutagenesis using positive antibiotic selection

Various mutsgenesis protocols have been established that use the hybridization of a mismatched oligonucleotide to prime DNA synthesis on an M13 phagemid template. For efficient mutagenesis, all of these methods require a means to select for the mutant strand before or during amplification in anEscherichia coli host. In the Altered Sites II protocol, the mismatched oligonucleotide and an oligonucleotide that restores antibiotic resistance to the phagemid are simultaneously hybridized to the template and coupled by DNA synthesis and ligation. The restored antibiotic resistance is then used to select only those phagemids which incorporate the antibiotic repair oligonucleotide. Generally, between 60 and 90% of the phagemids recovered will incorporate both oligonucleotides. This method provides a simple an efficient technique for introducing specific mutations into DNA.     

Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10–23 to enhance target affinity: Implications for DNAzyme design

Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg²⁺-dependent RNA-cleaving DNAzyme 10–23 (Dz10–23). As such, we have investigated the synthetic modification of Dz10–23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N′-biscyanoethoxycarbonyl)-guanidinoallyl-2′-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10–23. This particular modification was chosen as it had been used in the selection of Mg²⁺-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg²⁺ for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10–23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg²⁺-dependent DNAzyme with enhanced catalytic activity.     

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.     

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.     

Synthesis of oligonucleotide derivatives using ChemMatrix supports

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

Enzymatic synthesis of biphenyl-DNA oligonucleotides

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solid-phase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdT-mediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3′-end.     

Mass spectrometry of oligonucleotides

Expanding research in the field of modified oligonucleotides demands suitable analytical tools for size and purity verification of known compounds and accurate structure elucidation of unknowns. There is a need for characterization of the types and sites of modifications in oligonucleotides and to identify and sequence selected candidates originating from synthesis. The potential of electrospray tandem mass spectrometry (ESI-MS/MS) for structural characterization and sequencing of oligonucleotides is demonstrated. The fundamental behavior of DNA, RNA, and selected modified oligonucleotides in gas-phase is shown. Since gas-phase dissociation does not demand specific structural prerequisites, the method bears a great potential for rapid and most accurate characterization of modified oligonucleotides, e.g. from combinatorial libraries.