Solid-phase Oligonucleotide Synthesis Research Articles

Solid-Phase Synthesis of DNA and RNA 5'-O-Triphosphates Using cycloSal Chemistry.

A chemical method for the synthesis of short strands of DNA and RNA 5'-O-triphosphates is described that makes use of the conventional coupling and oxidizing reagents that are readily available on standard DNA/RNA synthesizers. After automated solid-phase synthesis of oligonucleotides and 5'-O-detritylation, a novel cycloSal-phosphoramidite, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite, was coupled to the 5'-hydroxyl group and the product oxidized. The resulting support-bonded 5'-cycloSal-oligonucleotide was reacted with pyrophosphate to yield 5'-O-triphosphorylated DNA/RNA oligonucleotides after cleavage from the support in high purity and excellent yields. The reaction sequence was adapted to be used completely on a standard automated oligonucleotide synthesizer. © 2016 by John Wiley & Sons, Inc.

Highly Ordered Pyrene π-Stacks on an RNA Duplex.

The syntheses of 2'-O-(pyren-1-ylmethyl)uridine phosphoramidite, 2'-O-(pyren-1-ylmethyl)adenosine phosphoramidite, and multiple pyrene-attached oligo-RNAs are described in this unit. The 2'-O-(pyren-1-ylmethyl)nucleosides are converted into the corresponding 2'-O-(pyren-1-ylmethyl)nucleoside 3'-phosphoramidites, which can be incorporated into the specific position of oligo-RNAs by solid-phase oligonucleotide synthesis. The multiple pyrene-attached oligo-RNA forms an A-form duplex with a complementary multiple pyrene-attached oligo-RNA; the pyrenes are associated with π-stacking along the outside of the duplex.

Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.

Native Chemical Ligation of Hydrolysis-Resistant 3'-NH-Cysteine-Modified RNA.

Hydrolysis-resistant RNA-peptide conjugates that contain a 3'-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3'-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3'-NH-peptide-modified RNAs, containing the universally conserved 3'-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle.

Duplex DNA and DNA-RNA hybrids with parallel strand orientation: 2'-deoxy-2'-fluoroisocytidine, 2'-deoxy-2'-fluoroisoguanosine, and canonical nucleosides with 2'-fluoro substituents cause unexpected changes on the double helix stability.

Oligonucleotides with parallel or antiparallel strand orientation incorporating 2'-fluorinated 2'-deoxyribonucleosides with canonical nucleobases or 2'-deoxy-2'-fluoroisocytidine ((F)iCd, 1c) and 2'-deoxy-2'-fluoroisoguanosine ((F)iGd, 3c) were synthesized. To this end, the nucleosides 1c and 3c as well as the phosphoramidite building blocks 19 and 23 were prepared and employed in solid-phase oligonucleotide synthesis. Unexpectedly, (F)iCd is not stable during oligonucleotide deprotection (55 °C, aq NH3) and was converted to a cyclonucleoside (14). Side product formation was circumvented when oligonucleotides were deprotected under mild conditions (aq ammonia-EtOH, rt). Oligonucleotides containing 2'-fluoro substituents ((F)iCd, (F)iGd and fluorinated canonical 2'-deoxyribonucleosides) stabilize double-stranded DNA, RNA, and DNA-RNA hybrids with antiparallel strand orientation. Unexpected strong stability changes are observed for oligonucleotide duplexes with parallel chains. While fluorinated oligonucleotides form moderately stable parallel stranded duplexes with complementary DNA, they do not form stable hybrids with RNA. Furthermore, oligoribonucleotide duplexes with parallel strand orientation are extremely unstable. It is anticipated that nucleic acids with parallel chains might be too rigid to accept sugar residues in the N-conformation as observed for ribonucleosides or 2'-deoxy-2'-fluororibonucleosides. These observations might explain why nature has evolved the principle of antiparallel chain orientation and has not used the parallel chain alignment.

Practical Synthesis of Cytidine-5-Carboxamide-Modified Nucleotide Reagents

Chemically-modified derivatives of cytidine, bearing a 5-(N-substituted-carboxamide) functional group, are new reagents for use in aptamer discovery via the SELEX process (Systematic Evolution of Ligands by EXponential enrichment). Herein, we disclose a practical synthesis of 5-(N-benzylcarboxamide)-2′-deoxycytidine, and the corresponding 5-(N-1-naphthylmethylcarboxamide)- and 5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine analogs, as both the suitably-protected 3′-O-cyanoethylphosphoramidite reagents (CEP; gram scale) and the 5′-O-triphosphate reagents (TPP; milligram-scale). The key step in the syntheses is a mild, palladium(0)-catalyzed carboxyamidation of an unprotected 5-iodo-cytidine. Use of the CEP reagents for solid-phase oligonucleotide synthesis was demonstrated and incorporation of the TPP reagents by KOD polymerase in a primer extension assay confirmed the utility of these reagents for SELEX. Finally, the carboxyamidation reaction was also used to prepare the nuclease-resistant sugar-variants: 5-(N-benzylcarboxamide)-2′-O-methyl-cytidine and 5-(N-3-phenylpropylcarboxamide)-2′-deoxy-2′-fluoro-cytidine.

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Synthesis, dynamic combinatorial chemistry, and PCR amplification of 3'-5' and 3'-6' disulfide-linked oligonucleotides.

Disulfide dithymidines linked 3'-5' or 3'-6' were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid-phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols and oligonucleotides. This link was shown to be sequence-adaptive in response to given templates in the presence of mercaptoethanol. The artificial 3'-5' and 3'-6' disulfide link was tolerated by polymerases in the polymerase chain reaction (PCR). By using sequencing analysis, we show that single mutations frequently occurred randomly in the amplification products of the PCR.

Independent generation and reactivity of uridin-2'-yl radical.

The uridin-2′-yl radical (1) has been proposed as an intermediate during RNA oxidation. However, its reactivity has not been thoroughly studied due to the complex conditions under which it is typically generated. The uridin-2′-yl radical was independently generated from a benzyl ketone (2a) via Norrish type I photocleavage upon irradiation at λmax = 350 nm. Dioxygen and β-mercaptoethanol are unable to compete with loss of uracil from 1 in phosphate buffer. Thiol trapping competes with uracil fragmentation in less polar solvent conditions. This is ascribed mostly to a reduction in the rate constant for uracil elimination in the less polar solvent. Hydrogen atom transfer to 1 from β-mercaptoethanol occurs exclusively from the α-face to produce arabinouridine. Mass balances range from 72 to 95%. Furthermore, the synthesis of 2a is amenable to formation of the requisite phosphoramidite for solid-phase oligonucleotide synthesis. This and the fidelity with which the urdin-2′-yl radical is generated from 2a suggest that this precursor should be useful for studying the radical’s reactivity in synthetic oligonucleotides.

Photogenerator of trichloroacetic acid as a promising detritylation agent for oligonucleotide microarray synthesis

[[4-(4-Methoxyphenyl)-2,6-dinitro-phenyl](phenyl)methyl]-2,2,2-trichloroacetate, which generates trichloroacetic acid when irradiated with light of 405 nm wavelength, has been studied as a detritylation agent in the oligonucleotide microarray synthesis. This agent was shown to be suitable for the deprotection of the 4,4′-dimethoxytrityl group during the solid phase oligonucleotide synthesis.

Synthesis of mixed-sequence oligonucleotides on mesoporous silicon: chemical strategies and material stability.

Rapid screening tests in medical diagnostic and environmental analysis are often based on oligonucleotide biochips. In this paper, we studied the stability of functionalized mesoporous silicon supports in the solid-phase synthesis of oligonucleotides, exploiting several chemical procedures. A 19-mer mixed sequence has been successfully synthesized on aminosilane-modified porous silicon photonic structures. The process and the materials have been characterized by optical reflectivity, atomic force microscopy and high-performance liquid chromatography.

Synthesis of cross-linked DNA containing oxidized abasic site analogues.

DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.

Efficient functionalization of oligonucleotides by new achiral nonnucleosidic monomers.

A novel synthetic strategy has been designed for preparation of achiral nonnucleosidic phosphoramidite monomers for automated solid-phase oligonucleotide synthesis. It is based on O-DMTr-protected 4-(2-hydroxyethyl)-morpholine-2,3-dione as the key compound and a family of building blocks obtained by its ring-opening by primary aliphatic amines. A series of nonnucleosidic phosphoramidites containing various side-chain functionalities was synthesized, and corresponding oligodeoxyribonucleotides incorporating modified units in single or multiple positions along the chain were prepared.

Detection of cyclin D1 mRNA by hybridization sensitive NIC–oligonucleotide probe

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dUTO, (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dUTO at various positions. dUTO-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840ng/μl). Additionally, dUT can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dUTO oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

DNA with 3′-5′-Disulfide Links-Rapid Chemical Ligation through Isosteric Replacement

Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA.1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid-phase synthesis of oligonucleotides containing 5'- or 3'-mercapto-dideoxynucleotides and their chemical ligations, yielding 3'-5'-disulfide bonds as a replacement for 3'-5'-phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half-lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3'-5' orientation of the activation site. The influence of temperature on the templated reaction is shown.

Utility of 5′-O-2,7-Dimethylpixyl for Solid-Phase Synthesis of Oligonucleotides Containing Acid-Sensitive 8-Aryl-Guanine Adducts

To study the structural and biological impact of 8-aryl-2'-deoxyguanosine adducts, an efficient protocol is required to incorporate them site-specifically into oligonucleotide substrates. Traditional phosphoramidite chemistry using 5'-O-DMT protection can be limiting because 8-aryl-dG adducts suffer from greater rates of acid-catalyzed depurination than dG and are sensitive to the acidic deblock conditions required to remove the DMT group. Herein we show that the 5'-O-2,7-dimethylpixyl (DMPx) protecting group can be used to limit acid exposure and improve DNA synthesis efficiency for DNA substrates containing 8-aryl-dG adducts. Our studies focus on 8-aryl-dG adducts with 8-substituents consisting of furyl ((Fur)dG), phenyl ((Ph)dG), 4-cyanophenyl ((CNPh)dG), and quinolyl ((Q)dG). These adducts differ in ring size and sensitivity to acid-promoted deglycosylation. A kinetic study for adduct hydrolysis in 0.1 M aqueous HCl determined that (Fur)dG was the most acid-sensitive (55.2-fold > dG), while (Q)dG was the most resistant (5.6-fold > dG). The most acid-sensitive (Fur)dG was chosen for optimization of solid-phase DNA synthesis. Our studies show that the 5'-O-DMPx group can provide a 4-fold increase in yield compared to 5'-O-DMT for incorporation of (Fur)dG into DNA substrates critical for determining adduct impact on DNA synthesis and repair.

Ethynyl Side Chain Hydration during Synthesis and Workup of “Clickable” Oligonucleotides: Bypassing Acetyl Group Formation by Triisopropylsilyl Protection

Clickable oligonucleotides with ethynyl residues in the 5-position of pyrimidines ((eth)dC and (eth)dU) or the 7-position of 7-deazaguanine ((eth)c(7)G(d)) are hydrated during solid-phase oligonucleotide synthesis and workup conditions. The side products were identified as acetyl derivatives by MALDI-TOF mass spectra of oligonucleotides and by detection of modified nucleosides after enzymatic phosphodiester hydrolysis. Ethynyl → acetyl group conversion was also studied on ethynylated nucleosides under acidic and basic conditions. It could be shown that side chain conversion depends on the nucleobase structure. Triisopropylsilyl residues were introduced to protect ethynyl residues from hydration. Pure, acetyl group free oligonucleotides were isolated after desilylation in all cases.

Spiropyran Photoswitches in the Context of DNA: Synthesis and Photochromic Properties

A new design is presented for the incorporation of spiropyran photoswitches into nucleic acids by oligonucleotide solid phase synthesis. This design enables interaction of the 6-nitrospirobenzopyran (NitroBIPS) photoswitch with the adjacent nucleobases and leaves the photochemical properties of NitroBIPS intact. UV/Vis spectroscopy and HPLC revealed that NitroBIPS incorporated into DNA consists of up to 40 % merocyanine in its thermal equilibrium and undergoes reversible switching between the photoisomeric spiropyran (SP) and merocyanin (MC) state by alternating excitation using visible light or heat for at least fifteen switching cycles. Exchanging the chromene part of NitroBIPS on the DNA level gives access to differently substituted spiropyran derivatives allowing the screening for spiropyrans with suitable properties in a straightforward manner. Thus, by incorporating the highly hydrolysis-stable pyrido-spiropyran derivative PyBIPS pure light-triggered reversible switching of a spiropyran in DNA has been realized for the first time. Therefore, this design represents a new useful platform for investigating the photochromic behavior of different spiropyran photoswitches in a nucleic acid environment and for using spiropyrans to induce light- or heat-triggered changes in conformations or in fluorescence quenching properties of oligonucleotides.

Solid-Phase Synthesis of DNA Chemical Sensor

Oligonucleotides are essential components of many applications in molecular biology. The synthesis chemistry is robust and commercial oligonucleotide synthesizers have taken advantage of the chemistry to provide oligonucleotides of high quality and purity. This paper established nucleic acid synthesis platform to carry out the synthesis of the labeled nucleic acid probes based on the DNA synthesizer and solid-phase synthesis technology. We chose to study the automated synthesis starting from DMT protected FAM labeled amidite attached to controlled pore glass (CPG) support and the standard trityl-off oligonucleotide synthesis cycle was performed, yielding the solid-supported oligonucleotide. The reported automated solid-phase oligonucleotide synthesis procedure successfully employs the common iterative synthesis, deblocking, activation, coupling, capping, oxidation, and isolation steps in standard oligonucleotide synthesis. The automated synthetic approach can also be applied to oligonucleotides of different length, composition of nucleotide, demonstrating the universality of the method. Moreover, the synthesis involved the use of commercially available, safe, stable, and inexpensive reagents, particularly advantageous and attractive for their use in automated solid-phase synthesis. The synthesis allows custom tailoring of their structure to the requirements of biological assays within hours, as opposed to traditional approaches that require weeks or months of work in the laboratory. Therefore it will become much easier to investigate biological interactions and optimize for objectives such as the receptor mediated targeting of oligonucleotides.

Mass Spectrometric Determination of Polyfunctionalized Nucleoside Phosphoramidites Using LSI/FAB

Nucleoside phosphoramidites (PAs) are the most widely used building blocks in the contemporary solid-phase synthesis of oligonucleotides. Accurate molecular weight measurements of such acid-labile compounds may be easily determined in a matrix system comprising triethanolamine (TEOA)-NaCl by liquid secondary ionization mass spectrometry (LSIMS) or fast atom bombardment mass spectrometry (FABMS), using a double-focusing mass spectrometer. The present method rapidly and easily measures the accurate MWs of various PAs as adduct ions [M+Na]⁺, affording molecular formulas in place of elemental analysis. Further, the intensities by LSIMS of [M+Na]⁺ were found to be enhanced to the highest degree by adjustment of the mole ratio of PA and NaCl, fixing the amount of TEOA. In addition, the effects of the metal ion in the matrix were investigated. The present method is a powerful tool for the mass spectral identification of polyfunctionalized nucleoside PAs.