Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background
journal contributionposted on 2020-09-23, 14:34 authored by Sean B. Yeldell, Linlin Yang, Jaehee Lee, James H. Eberwine, Ivan J. Dmochowski
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.
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model mRNA transcriptsoligonucleotide probephotoinduced biotinylationTIVA probe caginggel comigrationoligonucleotide-based techniquecaging controloptimized probevivo analysisFRET analysisbrain tissue slicesSuccessful applicationiterative modificationsOligonucleotide ProbeMinimal Background Messenger RNASingle Neuronsbackground bindingcaging performancedesign motivationsTIVA technique hingesdesign processVivo Analysiscell types