Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)†
| Autor: | Sean B. Yeldell, Brittani K. Ruble, Ivan J. Dmochowski |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Cell type Cell Oligonucleotides Biochemistry Article Transcriptome 03 medical and health sciences Single-cell analysis medicine Humans RNA Messenger Physical and Theoretical Chemistry Cells Cultured Nuclease biology Oligonucleotide Chemistry Gene Expression Profiling Organic Chemistry RNA Fibroblasts Molecular biology Cell biology Gene expression profiling 030104 developmental biology medicine.anatomical_structure Molecular Probes biology.protein Single-Cell Analysis |
| Popis: | Single cell transcriptomics provides a powerful discovery tool for identifying new cell types and functions as well as a means to probe molecular features of the etiology and treatment of human diseases, including cancer. However, such analyses are limited by the difficulty of isolating mRNA from single cells within biological samples. We recently introduced a photochemical method for isolating mRNA from single living cells, Transcriptome In Vivo Analysis (TIVA). The TIVA probe is a “caged” polyU : polyA oligonucleotide hairpin designed to enter live tissue, where site-specific activation with 405 nm laser reveals the polyU-biotin strand to bind mRNA in a target cell, enabling subsequent mRNA isolation and sequencing. The TIVA method is well suited for analysis of living cells in resected tissue, but has not yet been applied to living cells in whole organisms. Adapting TIVA to this more challenging environment requires a probe with higher thermal stability, more robust caging, and greater nuclease resistance. In this paper we present modifications to the original TIVA probe with multiple aspects of enhanced stability. These newer probes utilize an extended 22mer polyU capture strand with two 9mer polyA blocking strands (“22/9/9”) for higher thermal stability pre-photolysis and improved mRNA capture affinity post-photolysis. The “22/9/9 GC” probe features a terminal GC pair to reduce pre-photolysis interactions with mRNA by more than half. The “PS-22/9/9” probe features a phosphorothioated backbone, which extends serum stability from |
| Databáze: | OpenAIRE |
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