揭示 DNA 三联体组装:质谱法研究用于增强基因靶向的改良三重形成寡核苷酸

IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Sarveenah Chandrasegaran, Jack W Klose, Tara L Pukala
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引用次数: 0

摘要

脱氧核糖核酸三聚体在一系列涉及基因和转录调控的生物过程中具有潜在的作用。利用这些高阶结构形成体内靶基因的一个主要挑战是它们的低稳定性,这取决于许多因素,包括序列中碱基的长度和组成。在此,我们主要利用原生质谱法探索了不同的 DNA 碱基修饰,努力使三重形成寡核苷酸(TFO)与双链目标位点之间的结合更强。这些修饰还可用于克服基因组启动子区域双链序列中的嘧啶中断,从而扩展三重目标序列,用于抗基因疗法。使用具有单个嘧啶中断的模型序列,结果表明,与纯 DNA 和含 dSpacer 的 TFO 相比,含有锁定核酸碱基修饰的三聚体形成寡核苷酸具有更高的三聚体结合倾向。然而,这些系统的三聚体形成能力受到多个高阶组装体竞争性形成的限制。我们还研究了与铜绿假单胞菌基因组中特定基因靶点相对应的三聚体形成序列,含 LNA 的 TFOs 是唯一能利用这些序列形成三聚体的变体。这项研究表明,利用合成修饰的 TFOs 在体内形成三重聚合体具有潜在治疗应用的优势,并突出了原生质谱法在研究三重聚合体形成方面的优势。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Unraveling DNA Triplex Assembly: Mass Spectrometric Investigation of Modified Triplex Forming Oligonucleotides for Enhanced Gene Targeting.

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.

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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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