FRET‐MC : A fluorescence melting competition assay for studying G4 structures in vitro,Biopolymers

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FRET‐MC : A fluorescence melting competition assay for studying G4 structures in vitro
Biopolymers ( IF 3.2 ) Pub Date : 2020-12-24 , DOI: 10.1002/bip.23415
Yu Luo _{1,

2} , Anton Granzhan ₁ , Daniela Verga ₁ , Jean-Louis Mergny ₂

Affiliation

G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRET-melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆Tm ), while non-specific competitors (e.g., single- or double-stranded sequences) have little effect. Sixty-five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.

中文翻译：

FRET-MC：用于体外研究 G4 结构的荧光熔解竞争试验

G-四链体 (G4) 在生物学、分析化学和纳米技术中起着至关重要的作用。G4 结构的稳定性受 G 四重奏的数量、环的长度和位置、侧翼基序以及其他结构元素（如凸起、加帽碱基对或三联体）的影响。G4Hunter 或 Quadparser 等算法可以通过计算四重倾向得分来预测给定序列是否是 G4 倾向的；但是，仍然需要实验验证。我们之前已经证明，这种验证并不总是直接的，并且通常需要多种技术的组合来明确确定一个序列是否形成 G-四链体。在本文中，我们采用了众所周知的 FRET 熔解测定法来批量表征 G4，其中添加了待测试的序列，作为未标记的竞争者，到由双标记探针 (F21T) 和特定四链体配体组成的系统。PhenDC3 优于 TMPyP4，因为它对 G-四链体具有更好的选择性。在这种所谓的 FRET-MC（熔解竞争）分析中，G4 形成竞争者导致配体诱导的稳定效应 (∆Tm) 显着降低，而非特异性竞争者（例如，单链或双链序列） ) 影响不大。使用具有不同典型二级结构的 65 个已知序列来验证该测定，随后将其用于评估先前未表征的 8 个新序列。G4 形成竞争者导致配体诱导的稳定效应 (ΔTm) 显着降低，而非特异性竞争者（例如，单链或双链序列）几乎没有影响。使用具有不同典型二级结构的 65 个已知序列来验证该测定，随后将其用于评估先前未表征的 8 个新序列。G4 形成竞争者导致配体诱导的稳定效应 (ΔTm) 显着降低，而非特异性竞争者（例如，单链或双链序列）几乎没有影响。使用具有不同典型二级结构的 65 个已知序列来验证该测定，随后将其用于评估先前未表征的 8 个新序列。

更新日期：2020-12-24

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