Fluorophore-Quencher Interactions Effect on Hybridization Characteristics of Complementary Oligonucleotides.
Fluorophore-Quencher Interactions Effect on Hybridization Characteristics of Complementary Oligonucleotides.
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DOI：
10.1039/c9ay00584f
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发表时间：
2019-06
期刊：
影响因子：
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通讯作者：
Zackary A Zimmers;N. M. Adams;W. E. Gabella;F. Haselton
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文献类型：
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作者：
Zackary A Zimmers;N. M. Adams;W. E. Gabella;F. Haselton
核酸通常用荧光报告分子共价修饰以产生依赖于杂交状态的光学信号。设计这样的核酸报道分子涉及选择荧光团、猝灭剂和荧光猝灭设计。本报告概述了这些选择对DNA杂交特性的影响，通过检查六个荧光团在四个淬灭方案：淬灭剂分子从荧光团偏移0，5，或10个碱基，和核苷酸淬灭。与右手DNA相比，左手L-DNA的类似结合特性被评估以量化每个淬灭方案的效果。将这些结果应用于自适应PCR方法，该方法监测荧光标记的L-DNA作为反应中类似的未标记的D-DNA的哨兵。相对于未标记的寡核苷酸，所有测试的荧光团和淬灭方案将寡核苷酸对的退火温度增加了0.5至8.5 °C的值。具有最小增加（0.5 °C）的设计是具有FAM荧光团的正义链和具有从FAM偏移10个碱基的黑洞淬灭剂2的反义链。不抵消猝灭剂分子的相同设计导致退火温度偏移5 °C。使用基于这些L-DNA设计中的每一个的温度切换进行PCR，并且对于10碱基偏移设计，效率显著增加，其具有最小的退火温度偏移。这些结果突出了选择适当的荧光猝灭方案的核酸光学信号的重要性。
Nucleic acids are often covalently modified with fluorescent reporter molecules to create a hybridization state-dependent optical signal. Designing such a nucleic acid reporter involves selecting a fluorophore, quencher, and fluorescence quenching design. This report outlines the effect that these choices have on the DNA hybridization characteristics by examining six fluorophores in four quenching schemes: a quencher molecule offset from the fluorophore by 0, 5, or 10 bases, and nucleotide quenching. The similar binding characteristics of left-handed L-DNA were evaluated in comparison with right-handed DNA to quantify the effect of each quenching scheme. These results were applied to the Adaptive PCR method, which monitors fluorescently-labeled L-DNA as a sentinel for analogous unlabeled D-DNA in the reaction. All of the tested fluorophores and quenching schemes increased the annealing temperature of the oligonucleotide pairs by values ranging from 0.5 to 8.5 °C relative to unlabeled oligonucleotides. The design with the smallest increase (0.5 °C) was a sense strand with a FAM fluorophore and an anti-sense strand with Black Hole Quencher 2 offset by 10 bases from the FAM. An identical design that did not offset the quencher molecules resulted in a shift in annealing temperature of 5 °C. PCR was performed using temperature switching based on each of these L-DNA designs, and efficiency was significantly increased for the 10-base offset design, which had the smallest shift in annealing temperature. These results highlight the importance of selecting an appropriate fluorescence quenching scheme for nucleic acid optical signals.