Fluorophore‐binding RNA aptamers and their applications

Abstract

Why image RNA? Of all the biological molecules, RNA exhibits the most diverse range of functions. Evidence suggests that transcription produces a wide range of noncoding RNAs (ncRNAs), both short (e.g., siRNAs, miRNAs) and long (e.g., telomeric RNAs) that regulate many aspects of gene expression, including the epigenetic processes that underlie cell fate determination, polarization, and morphogenesis. All these functions are realized through the exquisite temporal and spatial control of RNA expression levels and the stability of specific RNAs within well-defined sub-cellular compartments. Given the central importance of RNA in dictating cell behavior via gene-related functions, there is a great demand for RNA imaging methods so as to determine the composition of the cellular 'transcriptome' and to acquire a complete spatial-temporal profile of RNA localization. Recent advances in fluorophore-binding RNA aptamers promise to provide exactly this knowledge, which can ultimately advance our understanding of cell function and behavior in conditions of health and disease, and in response to external stimuli. WIREs RNA 2016, 7:843-851. doi: 10.1002/wrna.1383 For further resources related to this article, please visit the WIREs website.

RNA Localization in Bacteria

“…The development of RNA aptamer based imaging methods provides another possibility of live-cell RNA imaging. This approach utilizes RNA aptamer sequences added to the transcript of interest and fluorogenic small molecules that can freely diffuse into the cell, and become fluorescent upon binding to the RNA aptamer (see also several recent reviews (17, 46–48)). While not quite suitable for live-cell imaging, earlier developments of malachite green (MG), thiazole orange (TO), and dimethyl indole red (DIR) aptamers, etc.…”

“…Despite their relatively broad applications in the area of metabolite sensing through engineering to various riboswitches (also see reviews (17, 46–48)), the use of RNA aptamers in single-molecule RNA imaging is still considered to be limited. This limited application of the aptamer-based method for single-molecule RNA imaging might be related to, e.g., the limited brightness of the tag, and the requirement for correct folding of the aptamer in vivo .…”

Live cell imaging of single RNA molecules with fluorogenic Mango II arrays

“…These data demonstrate that the fluorescence recovery of the Mango aptamers can be used to enhance the reconstruction of super-resolved images. Additionally, the improvement in resolution is not solely contained to the use of SIM, but also that the fluorogenic properties of Mango enable higher contrast imaging of RNAs as previously speculated 46 especially within the nucleus where nuclear localisation of FP-MS2 can preclude RNA detection at the single-molecule level.…”

Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

“…Additionally, the improvement in resolution is not solely contained to the use of SIM, but also that the fluorogenic properties of Mango enable higher contrast imaging of RNAs as previously speculated 46 The second, is a fluorogenic RNA aptamer that has been shown to enable RNA imaging in cells, but not at the single molecule level 26 . RNA Mango is likely compatible with these technologies while enabling direct RNA imaging at single molecule resolution without the need for fluorescent proteins.…”