胍-骨干寡核苷酸的自动合成与纯化。

Kacper Skakuj, Katherine E Bujold, Chad A Mirkin
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引用次数: 2

摘要

本方案描述了一种基于碘和碱作为温和偶联试剂的方法来合成脱氧核糖核胍(dng)-具有胍主链的寡脱氧核苷酸类似物。dng显示出独特的特性,如高细胞摄取,低毒性和抗核酸酶降解的稳定性增加,但由于需要有毒和迭代的人工合成方案,它们的发展受到阻碍。本文报道的新合成方法消除了对有毒的氯化汞和刺鼻的硫苯酚的需求,这些对以前的DNG合成方法至关重要,并将它们的合成转化为MerMadeTM 12自动寡核苷酸合成器。该方法可以在1 ~ 5 μmol的范围内全自动合成长度达20个碱基的DNG链,以及5'-DNG- dna -3'嵌合体。我们还提供了详细和易于理解的说明,以适应MerMadeTM 12寡核苷酸合成器,使DNG和DNA/RNA寡核苷酸的平行合成。由于DNG键改变了寡核苷酸的总体电荷,我们还描述了基于提取或制备级凝胶电泳的纯化策略,以产生具有不同长度和DNG数量的寡核苷酸,以及表征最终产物的方法。总的来说,这篇文章提供了合成,纯化和处理dng和混合电荷DNG-DNA寡核苷酸的概述。©2020 Wiley期刊有限责任公司基本协议1:制备用于胍偶联的MerMadeTM合成器基本协议2:在MerMadeTM合成器上合成DNG链基本协议3:使用制备乙酸尿素(AU)纯化DNG链基本协议4:使用MALDI-TOF MS表征DNG链基本协议5:使用AU表征DNG链PAGE支持协议1:合成引发剂功能化的CPG支持协议2:硫脲单体的合成。
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Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides.

This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.

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来源期刊
Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
期刊最新文献
Issue Information Immobilized Carbohydrates for Preparation of 3′-Glycoconjugated Oligonucleotides Mutation Analysis of L-Thymidine-Induced Replication Products Using a Restriction Enzyme–Mediated Assay The Sulfo-Click Reaction and Dual Labeling of Nucleosides Controlling Gene-Silencing with Azobenzene-Containing siRNAs (siRNAzos)
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