Open AccessOligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background
Author(s) -
Sean B. Yeldell,
Linlin Yang,
Jaehee Lee,
James Eberwine,
Ivan J. Dmochowski
Publication year - 2020
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.0c00499
Subject(s) - oligonucleotide , biotinylation , messenger rna , transcriptome , computational biology , in vivo , biology , rna , microbiology and biotechnology , biophysics , chemistry , gene expression , gene , biochemistry , genetics
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.