使用 Klenow DNA 聚合酶对 RNA 进行模板化 3ʹ 末端荧光标记

IF 1.6 Q2 MULTIDISCIPLINARY SCIENCES MethodsX Pub Date : 2024-08-28 DOI:10.1016/j.mex.2024.102925
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引用次数: 0

摘要

使用基于荧光的方法研究 RNA 结构-功能动态的一个长期挑战是如何将荧光团精确附着到结构化 RNA 分子上。尽管该领域取得了重大进展,但现有技术仍有局限性,尤其是在长结构 RNA 的 3ʹ 端标记方面。为了应对这一挑战,我们开发了一种化学酶法,利用 Klenow DNA 聚合酶来标记 RNA。在这种方法中:-Klenow DNA 聚合酶在 DNA 寡核苷酸模板的引导下,在 RNA 的 3ʹ 端添加氨基修饰的核苷酸。-对于高度结构化的 RNA,与 RNA 互补的 DNA 寡核苷酸会破坏预先存在的分子内 RNA 结构。这一方法的进步使得在各种 RNA 底物的 3′末端特异性地加入单个修饰核苷酸成为可能,而不论其长度或二级结构如何。该技术操作简便,只需对不同的 RNA 靶标进行最小程度的修改,因此可广泛应用于各类研究人员。这种方法有可能极大地改进功能化 RNA 的开发,使其应用于各种领域。
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Templated 3ʹ terminal fluorescent labeling of RNA using Klenow DNA polymerase

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3ʹ end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:

  • Klenow DNA polymerase adds an amino-modified nucleotide to the 3ʹ end of the RNA, guided by the DNA oligonucleotide template.

  • An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.

  • For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structures.

This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3′ terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.

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来源期刊
MethodsX
MethodsX Health Professions-Medical Laboratory Technology
CiteScore
3.60
自引率
5.30%
发文量
314
审稿时长
7 weeks
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