An Oligonucleotide Delivery Platform to Enable Assessment of Intracellular Transcripts in Live Cells by Flow Cytometry.,Cytometry Part A

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An Oligonucleotide Delivery Platform to Enable Assessment of Intracellular Transcripts in Live Cells by Flow Cytometry.
Cytometry Part A ( IF 2.5 ) Pub Date : 2020-06-26 , DOI: 10.1002/cyto.a.24174
Beverly Z Packard ₁ , James A Wrightson ₁ , Akira Komoriya ₁

Affiliation

The measurement of mRNA transcripts in live cells has been limited by inefficient delivery vehicles for oligonucleotides. Using a delivery platform which utilizes fluorophores capable of forming intramolecular H‐type excitonic dimers, we show that antisense oligonucleotides (ASOs) can be delivered across the plasma membrane directly into the cytosol without receptor mediation. With HIV infection of CD4⁺ lymphocytes as a model system, we quantitate the level of viral infection present in live single cells with flow cytometry by measuring the hybridization of ASOs to viral sequences; we then compare this measurement with a standard HIV analysis, that is, binding of an antibody against the HIV cell surface protein gp120. The nucleic acids delivery platform described herein also enables inhibition of HIV infection by addition of ASO constructs targeting sequences in the virus' highly conserved 5′‐untranslated region. Our analysis quantitates the level of inhibition by comparing both the MFI values and the mean fluorescence intensity as calculated by integration under each curve. Thus, a means for measuring intracellular transcripts at the live single cell level and the potential for delivery of a new class of antiviral agents is described. © 2020 International Society for Advancement of Cytometry

中文翻译：

一种寡核苷酸递送平台，可通过流式细胞术评估活细胞中的细胞内转录物。

活细胞中 mRNA 转录物的测量受到低效寡核苷酸传递载体的限制。使用能够形成分子内 H 型激子二聚体的荧光团的递送平台，我们表明反义寡核苷酸 (ASO) 可以穿过质膜直接递送到细胞质中，而无需受体介导。艾滋病毒感染 CD4 ⁺淋巴细胞作为模型系统，我们通过测量 ASO 与病毒序列的杂交，用流式细胞术定量活单细胞中存在的病毒感染水平；然后我们将此测量与标准 HIV 分析进行比较，即抗体与 HIV 细胞表面蛋白 gp120 的结合。本文所述的核酸递送平台还能够通过在病毒的高度保守的 5' 非翻译区中添加靶向序列的 ASO 构建体来抑制 HIV 感染。我们的分析通过比较 MFI 值和通过每条曲线下的积分计算的平均荧光强度来量化抑制水平。因此，描述了一种在活单细胞水平上测量细胞内转录物的方法，以及递送新型抗病毒剂的潜力。

更新日期：2020-06-26

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