Imaging RNA in a cell with Corn

Techniques to track and follow proteins inside living cells have vastly improved over the last decade, however comparable methods for tracking RNA have lagged behind. This is primarily due to the lack of intrinsically fluorescent RNA to match the expertise of the protein kind, like GFP (green fluorescent protein). Corn is one of the latest developed fluorophore-binding RNA aptamers that is suitable for quantifying RNA transcription. Song et al. have used it to analyse the temporal dynamics of RNA polymerase III transcription surpassing previous studies using Northern blots (1).

The need for Corn

RNA polymerase III (Pol III) is responsible for around 15% RNA transcription in a cell. This includes the production of tRNAs, small nucleolar RNAs and the 5S rRNA found in ribosomes. Since these RNA products are involved in mRNA processing and translation (see more about mRNA production here https://asheekeyscienceblog.com/2017/09/09/get-transcribed-in-style-the-ctd-that-everyones-talking-about/), the rate of Pol III can have drastic influence over the cell.

However, since Pol III transcripts are not translated (not used to make proteins), GFP and other protein reporters cannot be used to track their progress in the cell. RNA reporters, such as Corn, can be used instead.

Corn > Broccoli > Spinach

This is not an ordering of my food taste – I actually love spinach – but instead a biologists’ joke to name the RNA aptamers a food that’s colour matches the fluorescence of the RNA aptamer-fluorophore complex. For example, both Broccoli and Spinach aptamers bind green fluorophores, whilst the fluorophore is yellow with Corn.

Broccoli and Spinach are both RNA aptamers that bind 3,5-difluro-4-hydroxybenzylidene imidazolinone (DFHBI), causing a transition of DFHBI from its non-fluorescent state to its green fluorescent one. However, when the complex gets excited it readily photobleaches due to light-induced isomerisation of DFHBI from a cis to trans state preventing further fluorescence. Broccoli and Spinach are therefore not suitable to track long-term kinetics of the pol III RNA transcripts.

This is where Corn comes in. The RNA structure of Corn was meticulously mutated, tested and refined to suppress the non-radioactive decay pathway allowing for quantitative detection of RNA (as opposed to the qualitative information gained from using Broccoli or Spinach).

Corn doesn’t bind DFHBI, but instead binds 3,5-difluro-4-hydroxybenzylidene imidazolinone-2-oxime (DFHO) which resembles the RFP (red fluorescent protein) fluorophore (Figure 1(1)). Like DFHBI, DFHO also has low cytotoxicity and low background fluorescence, which allows it to be used to study RNA in live cells (1). Song’s team successfully used Corn to see the effect of mTOR inhibitors over time on pol III transcription. Their results showed heterogeneous transcription suppression in individual cells, highlighting the ability of Corn as a RNA reporter.

corn.png
Figure 1: A schematic representation of the RNA aptamer Corn binding the fluorophore DFHO

 

A Corn sandwich

To activate DFHO, Corn sandwiches the fluorophore in G-quadruplexes in a homodimeric fashion (2), with one G-quadruplex interacting from each aptamer (Figure 1(1)). G-quadruplexes are also used by Spinach making it a common motif in RNA aptamers.

The co-crystal structure of the Corn-DFHO complex has revealed that the homodimer interface is asymmetric (2). This can be exploited in future studies to develop mutants that only allow fluorescence as heterodimers – this can then be used to study co-expression of RNA transcripts (Figure 1 (2)).

However, if Corn is to be applied to quantify mRNA levels a monomeric form of Corn will need to be developed.

Further Reading

(1)  Song et al. Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nature Chem Bio (2017)

(2)  Warner et al. A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nature Chem Bio (2017)

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