IMAGEtags

Abstract

Cell communications are essential to the organization, development, and maintenance of multicellular organisms. Much of this communication involves changes in RNA transcription and is dynamic. Most methods for studying transcription require interrupting the continuity of cellular function by sacrificing the communicating cells and capturing gene expression information by periodic sampling of individual cells or the population. The IMAGEtag technology to quantify RNA levels in living cells, demonstrated here in yeast, allows individual cells to be tracked over time as they respond to different environmental cues. IMAGEtags are short RNAs consisting of strings of a variable number of tandem aptamers that bind small-molecule ligands. The aptamer strings can vary in length and in configuration of aptamer constituents, such as to contain multiples of the same aptamer or two or more different aptamers that alternate in their occurrence. A minimum effective length is about five aptamers. The maximum length is undefined. The small-molecule ligands are enabled for imaging as fluorophore conjugates. For each IMAGEtag, two fluorophore conjugates are provided, which are FRET pairs. When a cell expresses an RNA containing an IMAGEtag sequence, the aptamers bind their ligands and bring the fluorophores into sufficiently close proximity to allow FRET. The background fluorescence of both fluorophores is minimal in the FRET channel. These features endow IMAGEtags with the sensitivity to report on mRNA expression levels in living cells.

Aptamer-enabled uptake of small molecule ligands

“…Thus, DRAGIN activity might not be observed with an aptamer that is linked to a larger molecule that would be less mobile and have less direct access to the internal membrane surface. We tested this hypothesis using strings of five and seven tobramycin aptamers (TOB3A) that have been previously demonstrated to bind their tobramycin ligands in vivo 17 , 19 . The string of five tandem tobramycin aptamers binds tobramycin with a range of K d s between 4.3 and 46 μM compared with the 240 nM K d estimated for the singlemer TOB3A aptamer 19 .…”

“…Saccharomyces cerevisiae BY4735 were transformed with the identified yeast expression vectors as previously described 17 . The transformed cells were grown in SD-Uracil media in presence of 2% of glucose or raffinose for 19 h at 30 °C.…”