Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.,ACS Chemical Biology

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Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-09-09 , DOI: 10.1021/acschembio.0c00499
Sean B Yeldell ₁ , Linlin Yang ₁ , Jaehee Lee ₂ , James H Eberwine ₂ , Ivan J Dmochowski ₁

Affiliation

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell’s response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome in vivo analysis (TIVA). Successful application of the TIVA technique hinges upon its oligonucleotide probe remaining completely inert (or “caged”) to mRNA unless photoactivated. To improve the reliability of TIVA probe caging in diverse and challenging biological conditions, we applied a rational design process involving iterative modifications to the oligonucleotide construct. In this work, we discuss these design motivations and present an optimized probe with minimal background binding to mRNA prior to photolysis. We assess its caging performance through multiple in vitro assays including FRET analysis, native gel comigration, and pull down with model mRNA transcripts. Finally, we demonstrate that this improved probe can also isolate mRNA from single living neurons in brain tissue slices with excellent caging control.

中文翻译：

用于具有最小背景的单个神经元的体内转录组分析 (TIVA) 的寡核苷酸探针。

从单个细胞中分离的信使 RNA (mRNA) 可以产生强大的生物学见解，包括发现具有独特功能的新细胞类型以及可能预测细胞对各种治疗剂反应的标记。我们之前介绍了一种基于寡核苷酸的技术，用于在称为体内转录组的活细胞内进行位点选择性、光诱导生物素化和 mRNA 捕获分析 (TIVA)。TIVA 技术的成功应用取决于其寡核苷酸探针对 mRNA 保持完全惰性（或“笼罩”）除非被光激活。为了提高 TIVA 探针在多样化和具有挑战性的生物条件下的可靠性，我们应用了一个合理的设计过程，包括对寡核苷酸构建体进行迭代修改。在这项工作中，我们讨论了这些设计动机，并提出了一种优化的探针，在光解之前与 mRNA 的背景结合最小。我们通过多次体外评估其笼养性能分析包括 FRET 分析、天然凝胶复合和下拉模型 mRNA 转录本。最后，我们证明这种改进的探针还可以从脑组织切片中的单个活神经元中分离出 mRNA，并具有出色的笼控制。

更新日期：2020-10-17

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