Issue 47, 2017

Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)

Abstract

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 <1 h to at least 48 h, and also mediates uptake into cultured human fibroblasts.

Graphical abstract: Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)

Supplementary files

Article information

Article type
Paper
Submitted
20 Sep 2017
Accepted
12 Okt 2017
First published
13 Okt 2017

Org. Biomol. Chem., 2017,15, 10001-10009

Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)

S. B. Yeldell, B. K. Ruble and I. J. Dmochowski, Org. Biomol. Chem., 2017, 15, 10001 DOI: 10.1039/C7OB02353G

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements