MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures
MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures
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DOI：
10.1002/elan.201900690
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发表时间：
2020-01
期刊：
影响因子：
3
通讯作者：
M. Foguel;Angelica M. Balcarcel;M. Reed;Percy Calvo-Marzal;Y. Gerasimova;D. Kolpashchikov;K. Chumbimuni-Torres
中科院分区：
文献类型：
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作者：
M. Foguel;Angelica M. Balcarcel;M. Reed;Percy Calvo-Marzal;Y. Gerasimova;D. Kolpashchikov;K. Chumbimuni-Torres
通过杂交探针进行核酸分析的一个主要挑战是探针的紧密结合与折叠成稳定二级结构的核酸靶的序列选择性识别之间的折衷。我们一直在开发一种基于四向连接（4 WJ）的传感器，该传感器由固定在电极表面上的通用茎环（USL）探针和两个适配器链（M和F）组成。该传感器显示出在室温下对单碱基错配具有高度选择性，能够使用相同的USL探针检测多个靶标，并且具有改进的检测折叠核酸的能力。然而，一些核酸靶标，包括天然RNA，被折叠成非常稳定的二级和三级结构，这甚至可能对4 WJ传感器构成挑战。这项工作描述了一种新的传感器，命名为MVF，因为它使用三个探针站M，V和F，这进一步提高了4 WJ传感器与折叠目标的性能。与缺乏链V的4 WJ传感器相比，MVF传感器以-32.82 kcal/mol的计算折叠能量询问16 S rRNA NASBA扩增子，已证明信号与背景比提高了2.5倍。所提出的设计可用作分析折叠核酸（包括天然RNA）的一般策略。
One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of −32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.