Current Protocols in Nucleic Acid Chemistry最新文献

This article describes chemical synthesis of 2′-fluorinated Northern methanocarbacyclic (2′-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), and their incorporation into oligonucleotides by solid-supported synthesis. This synthesis starts from commercially available cyclopent-2-en-1-one to obtain the fluorinated carbocyclic pseudosugar intermediate (S.13), which can be converted to the uridine intermediate by condensation with isocyanate, followed by cyclization, and to adenine and guanine precursors by microwave-assisted reactions. All four 2′-F-NMC phosphoramidites are synthesized from S.13 in a convergent approach, and the monomers are used for synthesis of 2′-F-NMC-modified oligonucleotides. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Preparation of fluorinated carbocyclic pseudosugar intermediate

Basic Protocol 2: Preparation of 2′-F-NMC uridine and cytidine phosphoramidites

Basic Protocol 3: Preparation of 2′-F-NMC adenosine phosphoramidite

Basic Protocol 4: Preparation of 2′-F-NMC guanosine phosphoramidite

Basic Protocol 5: Synthesis of oligonucleotides containing 2ʹ-F-NMC

4-Cyanoindole-2ʹ-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2ʹ-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4

Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr)

Basic Protocol 3: Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP)

Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase

An efficient method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition (“click reaction”). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this conjugation method has to be adjusted to be compatible with the sulfur-containing groups. The method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me₂S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into “clickable ONs” by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me₂S. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Conjugation on solid support

Support Protocol: Removal of 4,4′-dimethoxytrityl group from amino linker

Basic Protocol 2: Removal of protecting groups and cleavage from solid support

Basic Protocol 3: HPLC purification

Cover: In Senthivelan et al. (http://doi.org/10.1002/cpnc.100), the image shows Preparation of 7-methylguanosine monophosphate 2.

This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5′-O-monophosphate (m⁷GMP), 7-methylguanosine 5′-O-diphosphate (m⁷GDP), 7-methyl-2′-deoxyguanosine 5′-O-triphosphate (m⁷2′dGTP), and 7-methylguanosine 5′-O-triphosphate (m⁷GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc.

Basic Protocol: Synthesis of 7-methylguanosine nucleotides.

Cover: In Yamamoto et al. (http://doi.org/10.1002/cpnc.99), the image shows molecular design of trivalent GalNAc ligands. (A) A conventional trivalent pyramidal structure, (B) our truncated-pyramidal structure, (C) a structure of tandemly-conjugated monovalent GalNAc units we developed earlier (Yamamoto, Sawamura, Wada, Harada-Shiba, & Obika, 2016).

Ligand-targeted drug delivery (LTDD) has emerged as an attractive option in the field of oligonucleotide drugs following the great success of N-acetylgalactosamine (GalNAc)–conjugated siRNA and antisense oligonucleotides. GalNAc is a well-known ligand of the asialoglycoprotein receptor (ASGPR), and is classified as a C-type lectin associated with the metabolism of desialylated glycoproteins. This article describes the synthesis of a non-nucleosidic monovalent GalNAc phosphoramidite—a useful reagent for facilitating the conjugation of GalNAc epitopes into oligonucleotides using DNA synthesizers—together with some important caveats. The monomeric GalNAc consists of three parts: (1) a GalNAc moiety, (2) a linker moiety, and (3) a trans-4-hydroxyprolinol (tHP) branch point. The GalNAc moiety and the tHP moiety are coupled via a condensation reaction to prepare the monovalent GalNAc phosphoramidite. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Synthesis of N-acetylgalactosamine ligand

Basic Protocol 2: Preparation of trans-4-hydroxyprolinol building block

Basic Protocol 3: Preparation of GalNAc phosphoramidite

By measuring a DNA polymerase incorporation reaction on a very short time scale (5 ms to 10 s) and with a high enzyme concentration, the isolated event of a single nucleotide incorporation can be analyzed. Practically, this is done using a quench-flow instrument, which allows the rapid mixing of the enzyme-primer/template with the nucleotide substrate, after which the reaction is quenched and analyzed. We describe how to titrate the enzyme active site, how to determine, via a scouting experiment, the rate-limiting step in the polymerization reaction, and how to measure the apparent k_pol, K_d(DNA), and K_d(N) using burst or single-turnover experiments. We include equations for the calculation of the processivity of the polymerase, its nucleotide incorporation specificity and preference, and the activation energy difference for the incorporation of an incorrect nucleotide. Data analysis is discussed, as this is an essential part of accurate data generation in kinetic analyses. © 2019 by John Wiley & Sons, Inc.

The N²-position of 2′-deoxyguanosine (N²-position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N²-dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N²-dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N²-dG damaged DNAs. This article describes a detailed protocol of the synthesis of N²-aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N²-dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N²-dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N²-position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc.

In this article, the earlier reported procedure for the synthesis of 2′-O-β-D-ribofuranosyl nucleosides was extended to the synthesis of 2′-O-α-D-ribofuranosyl adenosine, a monomeric unit of poly(ADP-ribose). It consists in condensation of a small excess of 1-O-acetyl-2,3,5-tri-O-benzoyl-α,β-D-arabinofuranose activated with tin tetrachloride with 3′,5′-O-tetra-isopropyldisiloxane-1,3-diyl-ribonucleosides in 1,2-dichloroethane. The following debenzoylation and silylation of arabinofuranosyl residue and inversion of configuration at C-2ʹʹ atom of arabinofuranosyl residue and final removal of silyl protective groups gave 2′-O-α-D-ribofuranosyl adenosine in overall 13% to 21% yield. © 2019 by John Wiley & Sons, Inc.