MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures,Electroanalysis

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MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures
Electroanalysis ( IF 2.7 ) Pub Date : 2020-01-27 , DOI: 10.1002/elan.201900690
Marcos V. Foguel ₁ , Angelica M. Balcarcel ₁ , Mark A. Reed ₁ , Percy Calvo‐Marzal ₁ , Yulia V. Gerasimova ₁ , Dmitry M. Kolpashchikov _{1,

2} , Karin Y. Chumbimuni‐Torres ₁

Affiliation

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.

中文翻译：

MVF传感器能够分析具有稳定二级结构的核酸

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标的序列选择性识别之间的折衷。我们正在开发一种基于四向连接（4WJ）的传感器，该传感器由固定在电极表面上的通用茎环（USL）探针和两根适配器链（M和F）组成。该传感器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探针检测多个目标，并具有提高的检测折叠核酸的能力。但是，某些核酸靶标（包括天然RNA）会折叠成非常稳定的二级和三级结构，即使对于4WJ传感器也可能构成挑战。这项工作描述了一个新的传感器，之所以称为MVF，是因为它使用了三个探针台M，V和F，从而进一步提高了带有折叠目标的4WJ传感器的性能。MVF传感器以−32.82 kcal / mol的折叠能量计算的16S rRNA NASBA扩增子的讯问表明，与缺乏链V的4WJ传感器相比，信噪比提高了2.5倍。拟议的设计可用于分析折叠核酸（包括天然RNA）的一般策略。

更新日期：2020-01-27

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