Current Protocols in Nucleic Acid Chemistry最新文献

A detailed protocol for preparation 3′-glycoconjugated oligonucleotides is described based on one-pot immobilization of 4,4′-dimethoxytrityl-protected carbohydrates to a solid support followed by on-support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post-synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate-modified supports using conventional phosphoramidite chemistry produces 3′-glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of 1-O-tert-butyldimethylsilyl-6-O-(4,4′-dimethoxytrityl)-β-D-glucose

Basic Protocol 2: Synthesis of 6-O-dimethoxytrityl-2,3,1′,3′,4′,6′-hexa-O-benzoylsucrose

Basic Protocol 3: Synthesis of 6″-O-dimethoxytrityl-N-trifluoroacetyl-protected aminoglycosides

Basic Protocol 4: Synthesis of 3-O-dimethoxytrityl-propyl β-D-galactopyranoside

Basic Protocol 5: Synthesis of trivalent N-acetyl galactosamine cluster

Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support

Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates

This article describes experimental and analytical procedures for evaluating the efficiency and fidelity of DNA replication containing mirror-image thymidine (L-T) in E. coli. The procedure involves construction of DNA recombinants containing a restriction enzyme (PstI) recognition site in which the L-T lesion is site-specifically located within the PstI recognition sequence (CTGCAG). The recombinants are transfected into DH5α cells. DNA is extracted, amplified, and cleaved into relatively short fragments using different combinations of restriction enzymes to facilitate electrophoretic analysis. Detailed explanations for the restriction enzyme–mediated assay for detection of mutagenic properties of mirror-image thymidine at a predetermined site are also presented. Advantages and limitations of the assay are discussed by comparing it to other techniques used for detecting lesion-induced mutation efficiency, and a troubleshooting guide is provided. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of oligonucleotides containing L-T

Basic Protocol 2: Construction of DNA recombinants

Basic Protocol 3: Mutation analysis of L-T-induced replication products using a restriction enzyme–mediated assay

This article contains detailed synthetic procedures for the implementation of the sulfo-click reaction to nucleoside derivatives. First, 3′-O-TBDMS-protected nucleosides are converted to their corresponding 4′-thioacid derivatives in three steps. Then, various conjugates are synthetized via a biocompatible and chemoselective coupling procedure using sulfonyl azide partners. Finally, to illustrate the potential of the sulfo-click reaction, a nucleoside bearing two orthogonal azido groups is synthesized and engaged in one-pot dual labeling through a sulfo-click/copper-catalyzed azide-alkyne cycloaddition (CuAAC) cascade. The high efficiency of the sulfo-click reaction as applied to nucleosides opens up new possibilities in the context of bioconjugation. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: General protocol for the synthesis of 4′-thioacid-nucleoside derivatives

Basic Protocol 2: Implementation of the sulfo-click reaction

Basic Protocol 3: Synthesis of 3′-azido-4′-(carboxamido)ethane-sulfonyl azide-3′-deoxythymidine

Basic Protocol 4: Detailed synthetic procedure for one-pot double-click conjugations

This article contains the detailed biophysical characterization, biological testing, and photo-switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti-sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (T_m) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re-activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Bio-physical characterization of siRNAzo duplexes

Basic Protocol 2: Evaluation of azobenzene gene-silencing using Firefly Luciferase

Basic Protocol 3: Evaluation of azobenzene gene-silencing using reverse transcriptase-polymerase chain reaction (RT-PCR)

Tandem catalysis has been at the forefront of synthesis in the past decade due to the reduction in the number of steps and purification needed for the synthesis of commercially relevant molecules. With the right combination of catalyst systems, which could be homometallic or multimetallic, one can construct complex structural motifs in a one-pot procedure without the requirement for the isolation of the intermediates, reducing both reagent waste and time. Over the years, application of tandem catalysis has certainly extended towards arene and heteroarene motifs; nucleoside modification using such a strategy has been rare. In this regard, we would like to report herein the development of numerous homometallic and multimetallic tandem catalytic protocols for the modification of nucleosides, providing efficient access to a diverse range of molecules with promising fluorescent properties, as well as pharmaceutically relevant antiviral drugs such as FV-100. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Double tandem one-pot Sonogashira/cyclization of 5-IdU for the synthesis of FV-100 and analogs

Basic Protocol 2: Double tandem one-pot Heck/Suzuki–Miyaura of 5-IdU for the synthesis of fluorescent nucleoside analogs

Basic Protocol 3: Double tandem one-pot Suzuki–Miyaura cross-coupling of 5-IdU for the synthesis of fluorescent nucleoside analogs

Basic Protocol 4: Double tandem one-pot amination/amidation for the synthesis of Sangivamycin precursor

Basic Protocol 5: Triple tandem one-pot chemoselective etherification/Sonogashira coupling/cyclization for synthesis of BCNA analogs

Basic Protocol 6: Triple tandem one-pot sequential Heck/borylation/Suzuki-Miyaura reaction

The protocols presented in this article describe highly detailed synthesis of trifluoromethylated purine nucleotides and nucleosides (G and A). The procedure involves trifluoromethylation of properly protected (acetylated) nucleosides, followed by deprotection leading to key CF₃-containing nucleosides. This gives synthetic access to 8-CF₃-substituted guanosine derivatives and three adenosine derivatives (8-CF₃, 2-CF₃, and 2,8-diCF₃). In further steps, phosphorylation and phosphate elongation (for selected examples) result in respective trifluoromethylated nucleoside mono-, di-, and triphosphates. Support protocols are included for compound handling, purification procedures, analytical sample preparation, and analytical techniques used throughout the performance of the basic protocols. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of trifluoromethylated guanosine and adenosine derivatives

Basic Protocol 2: Synthesis of trifluoromethylated guanosine and adenosine monophosphates

Basic Protocol 3: Synthesis of phosphorimidazolides of ^8-CF3GMP and ^8-CF3AMP

Basic Protocol 4: Synthesis of trifluoromethylated guanosine and adenosine oligophosphates

Support Protocol 1: TLC sample preparation and analysis

Support Protocol 2: Purification protocol for Basic Protocol 1

Support Protocol 3: HPLC analysis and preparative HPLC

Support Protocol 4: Ion-exchange chromatography

NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope-labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear-detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope-labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo-enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear-detected NMR experiments including ¹³C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the ¹⁵N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment. © 2020 The Authors.

Basic Protocol 1: Preparation of isotope-labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP-HPLC purification

Alternate Protocol 1: Purification of isotope-labeled RNA from in vitro transcription with preparative PAGE

Alternate Protocol 2: Purification of isotope-labeled RNA samples from in vitro transcription via centrifugal concentration

Support Protocol 1: Preparation of DNA template from plasmid

Support Protocol 2: Preparation of PCR DNA as template

Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP)

Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP)

Basic Protocol 2: Preparation of site-specific labeled RNAs using a chemo-enzymatic synthesis

Support Protocol 5: Synthesis of modified nucleoside 3′,5′-bisphosphates

Support Protocol 6: Preparation of T4 RNA Ligase 2

Support Protocol 7: Setup of NMR spectrometer for heteronuclear-detected NMR experiments

Support Protocol 8: IPAP and DIPAP for homonuclear decoupling

Basic Protocol 3: ¹³C-detected 3D (H)CC-TOCSY, (H)CPC, and (H)CPC-CCH-TOCSY experiments for ribose assignment

Basic Protocol 4: ¹³C-detected 2D CN-spin filter HSQC experiment

Basic Protocol 5: ¹³C-detected C(N)H-HDQC experiment for the detection of amino groups

Support Protocol 9: ¹³C-detected CN-HSQC experiment for amino groups

Basic Protocol 6: ¹³C-detected “amino”-NOESY experiment

Basic Protocol 7: ¹⁵N-detected BEST-TROSY experiment

Custom-built DNA nanostructures are now used in applications such as biosensing, molecular computation, biomolecular analysis, and drug delivery. While the functionality and biocompatibility of DNA makes DNA nanostructures useful in such applications, the field faces a challenge in making biostable DNA nanostructures. Being a natural material, DNA is most suited for biological applications, but is also easily degraded by nucleases. Several methods have been employed to study the nuclease degradation rates and enhancement of nuclease resistance. This protocol describes the use of gel electrophoresis to analyze the extent of nuclease degradation of DNA nanostructures and to report degradation times, kinetics of nuclease digestion, and evaluation of biostability enhancement factors. © 2020 Wiley Periodicals LLC.

Basic Protocol: Timed analysis of nuclease degradation of DNA nanostructures

Support Protocol: Calculating biostability enhancement factors

This article contains detailed synthetic protocols for preparation of 5-cyanomethyluridine (cnm⁵U) and 5-cyanouridine (cn⁵U) phosphoramidites. The synthesis of the cnm⁵U phosphoramidite building block starts with commercially available 5-methyluridine (m⁵C), followed by bromination of the 5-methyl group to install the cyano moiety using TMSCN/TBAF. The cn⁵U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5-cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA-based therapeutics. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of cnm⁵U and cn⁵U phosphoramidites

Basic Protocol 2: Synthesis, purification, and characterization of cnm⁵U- and cn⁵U-modified RNA oligonucleotides

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides

Basic Protocol 2: Digestion of RNA samples extracted from cells

Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry