Current Protocols in Nucleic Acid Chemistry最新文献

This unit describes postsynthetic modification of oligonucleotides (ONs) containing 2′-deoxy-5-trifluoromethyluridine and 2′-deoxy-5-trifluoromethylcytidine. In ONs, the trifluoromethyl group at the C5 position of pyrimidine bases is converted into a variety of carboxylic acid equivalents using alkaline and amine solutions. In addition, treating fully protected and controlled pore glass (CPG)-attached ONs with methylamine and sodium hydroxide aqueous solution results in deprotection of all protecting groups (except the 4,4′-dimethoxytrityl group), cleavage from CPG, and simultaneous conversion of the trifluoromethyl group to afford the corresponding ONs containing 5-substituted pyrimidine bases. © 2019 by John Wiley & Sons, Inc.

This chemoenzymatic procedure describes a strategy for the preparation of 4'-thioribose nicotinamide adenine dinucleotide (S-NAD⁺ ), including chemical synthesis of nicotinamide 4'-riboside (S-NR), recombinant expression and purification of two NAD⁺ biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S-NAD⁺ . The first basic protocol describes the procedures for introduction of nicotinamide onto 4'-thioribose and subsequent deprotection to generate S-NR as the key intermediate for enzymatically synthesizing S-NAD⁺ . In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S-NR to S-NAD⁺ . The third basic protocol presents the enzymatic approach for the generation of S-NAD⁺ from S-NR precursor. © 2019 by John Wiley & Sons, Inc.

Immobilization of DNA is an important step in relation to DNA-based biosensors and bioassays with multiple applications. This unit describes synthesis and applications of novel bifunctional linker molecules containing nitrogen mustard and one of two types of functional groups: cyclic disulfide or biotin. Two ways of immobilizing DNA on a surface are described. With the first method, a bifunctional alkylating linker molecule is first reacted with the target DNA to form alkylated DNA and then immobilized on a specific surface. With the second method, the bifunctional alkylating linker molecule is first attached to the surface, and then the target DNA is immobilized through an alkylating reaction with a nitrogen mustard moiety. We have also achieved immunochemical detection and quantification of 5-methylcytosine in a target DNA immobilized by the above methods. The methods for immobilization of intact DNA using novel bifunctional linker molecules are applicable to a wide range of biological analysis techniques. © 2019 by John Wiley & Sons, Inc.

Nucleic acid triplexes are formed when a DNA or RNA oligonucleotide binds to a polypurine-polypyrimidine-rich sequence. Triplexes have wide therapeutic applications such as gene silencing or site-specific mutagenesis. In addition, protocols based on triplex-affinity capture have been used for detecting nucleic acids in biosensing platforms. In this article, the design, synthesis, and use of parallel clamps and polypurine-reversed hairpins (PPRH) to bind to target polypyrimidine targets are described. The combination of the polypurine Watson-Crick strand with the triplex-forming strand in a single molecule produces highly stable triplexes allowing targeting of single- and double-stranded nucleic acid sequences. On the other hand, PPRHs are easily prepared and work at nanomolar range, like siRNAs, and at a lower concentration than that needed for antisense ODNs or TFOs. However, the stability of PPRHs is higher than that of siRNAs. In addition, PPRHs circumvent off-target effects and are non-immunogenic. © 2019 by John Wiley & Sons, Inc.

This unit describes the detailed preparation of 5-alkynyl-2'-halogenated arabinosyl uridine nucleosides (2'-halo-ara-EdU) from uridine. These compounds were synthesized as prospective chemical probes for the detection of DNA synthesis in proliferating cells. Currently, this is the only synthetic methodology reported to access these compounds. The key to success of the synthetic approach was to employ a 3-N-nitro-protecting group to stabilize the required 2'-triflate nucleoside precursor toward nucleophilic substitution. Several synthetic challenges were overcome to accommodate the combination of a 5-alkyne and 3-N-nitro functional group, including facile introduction and removal of the N-nitro group, and removal of the sugar acetyl groups under acidic conditions. © 2019 by John Wiley & Sons, Inc.

Straightforward and efficient methods for the synthesis of 2-amino-4-fluoropyridine-C-nucleoside (d^F P) and the solid-phase synthesis of oligodeoxynucleotides containing d^F P using a phosphoramidite are described. The synthesis of d^F P is achieved by cross-coupling between a nucleobase (2-amino-4-fluoro-3,5-diiodopyridine) and sugar moieties. Its 3'-O-phosphoramidite is obtained by deiodination, 5'-O-protection, and 3'-O-phosphitilation in three steps. The phosphoramidite unit is compatible for the synthesis of oligonucleotides on solid-phase according to conventional phosphoramidite chemistry. The 2-amino-4-fluoropyridine-C-nucleoside moiety incorporated into the oligodeoxynucleotide reacts with a Cys residue in the catalytic site of DNA cytosine-5-methyltransferase (DNMT). It is apparent that 2-amino-4-fluoropyridine-C-nucleoside would be utilized in DNA-protein crosslink technology. This protocol describes the importance of solid-phase synthesis to obtain novel pyridine-C-nucleoside analogues and its incorporation into oligodeoxynucleotides in a short period of time. © 2019 by John Wiley & Sons, Inc.

This article describes procedures to synthesize 2'-OMe-RNA modified with cross-linkable 2-amino-7-deaza-7-propynyl-6-vinylpurine (ADpVP) and preparation of the RNA-crosslinking experiment in vitro. All synthesis steps yield the desired compound in moderate or high yield without expensive chemical reagents or specific devices. The crosslink-active form of modified RNA can also be purified by commonly used reversed-phase HPLC, can be stored at -80°C after lyophilization for a few days, and is ready to use for crosslinking experiments. This crosslink-active RNA can efficiently form covalent bonds with complementary RNA in a sequence-specific manner. © 2019 by John Wiley & Sons, Inc.

This unit describes the detailed synthetic protocol for the preparation of the phosphoramidite units of the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These C-nucleoside derivatives are useful units for the incorporation into triplex forming oligonucleotides (TFOs) to form the stable triplex DNA containing the CG interrupting sites. Commercially available 1-methyl-2'-deoxypseudouridine is prepared from thymidine and 5-iodo-uracil by a simple method, that is, coupling of glycal and 5-iodo-1-methyluracil by the Heck reaction, followed by desilylation and diastereoselective reduction. The carbonyl group at the 4 position of the pseudouridine derivative is activated by 3-nitorotriazole and treated with the corresponding aromatic amine compounds to produce the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These derivatives are then successfully converted to the phosphoramidite units and incorporated into the oligodeoxynucleotides. © 2019 by John Wiley & Sons, Inc.

Therapeutic oligonucleotides have emerged as a powerful paradigm with the ability to treat a wide range of the human diseases. As a result, we have witnessed more than one hundred oligonucleotides currently in active clinical trials and eight Food and Drug Administration (FDA)-approved drugs. Until now, the demand for oligonucleotide-based drugs has been fulfilled by conventional solid-phase synthesis in an effective manner. However, there are products in advanced stages of clinical trials projecting a collective demand of metric ton quantities in the near future. Therefore, large-scale manufacturing of these products has become a high priority for process chemists. This article summarizes the advances in liquid-phase oligonucleotide synthesis (LPOS) as a possible alternative strategy to meet the scale-up challenge. A review of the literature describing major efforts in developing LPOS technologies is presented. Gratifyingly, serious attempts are under way to develop an efficient environmentally benign green chemistry protocol that is scalable and cost effective for the manufacturing of oligonucleotides. A summary of the most innovative LPOS protocols has been included to provide a glimpse of what may be possible in the future for large-scale production of oligonucleotides. © 2019 by John Wiley & Sons, Inc.