Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

Abstract

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitability as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…PFP-DFHBI exhibits 5-fold higher binding affinity to Spinach2 than DFHBI. Therefore, Spinach2-PFP-DFHBI shows 3–4-fold brighter fluorescence and less salt concentration dependency 58 . TO1 can greatly enhance its fluorescence by binding to G4 quadruplexes containing aptamers.…”

“…(b) Chemical structures of fluorophores in the RNA aptamer/fluorophore systems: DFHBI, DFHBI-1T 57 , PFP-DFHBI 58 , TO1 56 .…”

IMAGEtags

“…4) The aptamers should be tested for their abilities to bind ligand under similar conditions as present in the cytoplasm in which the free Mg 2+ concentration is much lower than is used for selecting most aptamers and where the major monovalent cation is K + rather than Na + . We have found that aptamer affinities and ligand specificities can be quite different in buffers that approximate intracellular conditions compared with the buffers in which they were selected (Muslum Ilgu, et al, 2016; M. Ilgu, Wang, Lamm, & Nilsen-Hamilton, 2013).…”

“…Instead, we created IMAGEtags consisting of multiple copies of the same aptamer sequence in strings of at least five units. These IMAGEtags function well for imaging promoter activity when the aptamer ligand is presented in two forms, each conjugated with one of a FRET pair (Muslum Ilgu et al, 2016; Shin et al, 2014). The effectiveness of these single-aptamer IMAGEtags can be understood when it is considered that, when an internal aptamer of the string binds one of the two ligands, there is a 50% chance that one or the other adjacent aptamer will bind the FRET pair and therefore a 75% chance that the central aptamer will have an adjacent FRET partner.…”

“…Thus, an IMAGEtag containing a single aptamer type will provide a signal that increases in efficiency with the number of tandem aptamers in the string. We have found that strings of 5 aptamers or more of a single type provide sufficient signal to detect transcribed mRNA (Muslum Ilgu, et al, 2016; Shin, et al, 2014). …”