Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis
Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis
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DOI：
10.1039/d1an01802g
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发表时间：
2022-01-27
期刊：
影响因子：
4.2
通讯作者：
Wong，Pak Kin
中科院分区：
文献类型：
--
作者：
Mercedes，Samuel A. Vilchez;Eder，Ian;Wong，Pak Kin
双链生物传感器是一种同质寡核苷酸探针，用于生物化学分析和活细胞成像中的核酸序列检测。锁核酸（LNA）修饰可以并入生物传感器中以增强结合亲和力、特异性和对核酸酶降解的抗性。然而，猝灭剂序列中的LNA单体也可以阻止靶-荧光团探针结合，这降低了dsLNA生物传感器的信号。本研究通过改变猝灭剂序列中存在的LNA单体的位置和量来研究LNA修饰对dsLNA生物传感器的影响。我们表征了荧光团-猝灭剂相互作用、目标检测和生物传感器在游离溶液中的特异性，并评估了dsLNA生物传感器在2D单层和3D球状体中的性能。数据表明，淬灭剂序列中的大量LNA单体可以增强生物传感器的特异性，但阻止有效的靶结合。总之，我们的研究结果为提高dsLNA生物传感器在活细胞核酸检测和基因表达分析中的性能提供了指导。
Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore–quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.