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

Date

2016-04-01

Author

İlgü, Müslüm
BENDICKSON, Lee
WANG, Tianjiao
Geraskin, Ivan M.
Kraus, George A.
Nilsen-Hamilton, Marit

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

126
views

0
downloads

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 suitabilities 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.

Subject Keywords

General Biochemistry, Genetics and Molecular Biology, Molecular Biology

URI

https://hdl.handle.net/11511/38902

Journal

METHODS

DOI

https://doi.org/10.1016/j.ymeth.2015.12.009

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Investigating the malleability of RNA aptamers İlgü, Müslüm; Lamm, Monica H.; Nilsen-Hamilton, Marit (Elsevier BV, 2013-09-15) Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemic...
The NF-kappa B target genes ICAM-1 and VCAM-1 are differentially regulated during spontaneous differentiation of Caco-2 cells Astarci, Erhan; Sade, Asli; Cimen, Ismail; SAVAŞ, BERNA; Banerjee, Sreeparna (Wiley, 2012-08-01) Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cellcell and cellmicroenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line, which undergoes spontaneous re-differentiation when grown past confluency, we observed a loss of VCAM-1 (vascular cell adhesion molecule 1) mRNA expression, while ICAM-1 (intercellular cell adhesion m...
Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex Zhang, Heng; Devoucoux, Maëva; Song, Xiaosheng; Li, Li; Ayaz, Gamze; Cheng, Harry; Tempel, Wolfram; Dong, Cheng; Loppnau, Peter; Côté, Jacques; Min, Jinrong (Elsevier BV, 2020-3) MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we p...
The Karyote® Physico-Chemical Genomic, Proteomic, Metabolic Cell Modeling System Ortoleva, P.; Berry, E.; Brun, Y.; Fan, J.; Fontus, M.; Hubbard, K.; Jaqaman, K.; Jarymowycz, L.; Navid, A.; Sayyed-Ahmad, A.; Shreif, Z.; Stanley, F.; Tuncay, Kağan; Weitzke, E.; Wu, L.-C. (Mary Ann Liebert Inc, 2003-01-01) Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical model...
Immunotherapeutic applications of CpG ODN Gürsel, Mayda (2006-06-01) Bacterial DNA and synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs stimulate the mammalian immune system to mount a rapid innate immune response. This response is characterized by the production of polyreactive IgM, immunomodulatory cytokines and chemokines. CpG ODN directly stimulate lymphocytes, natural killer cells and professional antigen-presenting cells (such as macrophages and dendritic cells). Owing to the strength and nature of this stimulation, CpG ODN are being harnessed f...

Citation Formats

M. İlgü, L. BENDICKSON, T. WANG, I. M. Geraskin, G. A. Kraus, and M. Nilsen-Hamilton, “Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells,” METHODS, pp. 26–33, 2016, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/38902.