Self-Assembly of Intracellular Multivalent RNA Complexes Using Dimeric Corn and Beetroot Aptamers

Abstract

DNA and RNA can spontaneously self-assemble into various structures, including aggregates, complexes, and ordered structures. The self-assembly reactions cannot be genetically encoded to occur in living mammalian cells since the double-stranded nucleic acids generated by current self-assembly approaches are unstable and activate innate RNA immunity pathways. Here, we show that recently described dimeric aptamers can be used to create RNAs that self-assemble and create RNA and RNA–protein assemblies in cells. We find that incorporation of five copies of Corn, a dimeric fluorogenic RNA aptamer, into an RNA causes the RNA to form large clusters in cells, reflecting multivalent RNA–RNA interactions enabled by these RNAs. Here, we also describe a second dimeric fluorogenic aptamer, Beetroot, which shows partial sequence similarity to Corn. Both Corn and Beetroot form homodimers with themselves but do not form Corn–Beetroot heterodimers. We thus use Corn and Beetroot to encode distinct RNA–protein assemblies in the same cells. Overall, these studies provide an approach for inducing RNA self-assembly, enable multiplexing of distinct RNA assemblies in cells, and demonstrate that proteins can be recruited to RNA assemblies to genetically encode intracellular RNA–protein assemblies.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…Compared to these tools, RNA aptamers: SFs systems exhibit high fluorogenic activity and In addition to these five systems that were used for single mRNA imaging, there are other reported RNA aptamers: SFs systems. For example, we and Jaffrey lab reported Corn: DFHO, [34] Beetroot: DFAME, [35] Broccoli: TBI, [36] Red Broccoli: OBI, [37] Squash: DFHO [38] systems recently. Höbartner lab reported Chili: DMHBI system.…”

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging

“…Since Beetroot and Corn both form a homodimer, they have been used as tandem repeat tags in live-cell imaging to induce macromolecular assembly via multivalent intermolecular interaction. 59 Together with fluorescent proteins, the authors also achieved orthogonal multi-colour imaging of RNA-protein assemblies in HEK293T cells simultaneously.…”

“…3C, highlighted in orange oval) has been used to visualize orthogonal RNA-protein assemblies in cells simultaneously. 59 Since both Beetroot and Corn aptamers are homodimers, they enable the formation of orthogonal macromolecular assemblies through dimerization interactions upon tagging multiple copies of each aptamer on the ROI. These RNA assemblies can not only be visualized by the fluorescence of RNA tags, but can also form larger RNA-protein assemblies by recruiting specific protein partners upon the combined use of the MS2-MCP and boxB-N-peptide systems.…”

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

“…Li et al described a light-up Spinach-based cascade transcriptional amplification strategy for ultrasensitive and label-free detection of miRNAs, opening a new avenue for the construction of RNA aptamer-based fluorescent biosensors. Corn is a 28-nucleotide fluorescent light-up RNA aptamer, and it can specifically bind with small-molecule fluorogens 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) to activate its fluorescence. , In comparison with Spinach, Mango, and Malachite Green chromophores, the Corn–DFHO complex exhibits a small size, low background, minimal cytotoxicity, and enhanced photostability . To the best of our knowledge, the use of Corn–DFHO complex as the signal indicator for lncRNA detection has not been reported so far.…”

“…Corn is a 28-nucleotide fluorescent light-up RNA aptamer, and it can specifically bind with small-molecule fluorogens 3,5difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) to activate its fluorescence. 28,29 In comparison with Spinach, 30 Mango, 31 and Malachite Green chromophores, 32 the Corn−DFHO complex exhibits a small size, low background, minimal cytotoxicity, and enhanced photostability. 33 To the best of our knowledge, the use of Corn−DFHO complex as the signal indicator for lncRNA detection has not been reported so far.…”