RNA TOOLS

Nucleic acids play many important roles in cells making their detection important both in vivo, for biological study, and in vitro for RNA diagnostics. Tracking RNA molecules in real time is a challenging problem as RNA is not intrinsically fluorescent. To address this problem, we use in vitro selection to identify high affinity flurogenic aptamers, which dramatically enhance the fluorescence of fluorogenic ligands upon binding.

RNA mANGO

We have selected fluorogenic RNA aptamers, called RNA Mango, that bind tightly to small cell permeable thiazole orange type ligands (Dolgosheina et al. 2014). These fluorogenic ligands becomes thousands of times brighter upon being bound, becoming brighter than eGFP. As binding affinities are in the low nanomolar range, the fluorescence efficiency of these fluorescent systems is very high, giving them high contrast. Due to their high brightness and binding affinity these aptamers have many uses, ranging from single molecule imaging in live cells (Cawte et al. 2020) to RNP pulldowns (Panchapakesan et al. 2017).

 

cHAraCTERIZATION AND dEVELOPMENT OF rna maNGO

Structure Guided Engineering

The structures of the RNA Mango aptamer series (I, II, III, and IV) have been solved by X-ray crystallography in collaboration with the Ferré D’Amaré lab. By using these crystal structures as a reference, we have been able to successfully identify and modify nucleotides of interest. In Mango III, for example, we have identified two nucleotides that base pair and that stack directly on top of TO1-B binding pocket. Through structure guided engineering of this two-nucleotide cap, we found a superior variant Mango III A10U that is 114% of its total brightness to its wild type counterpart (Trachman et al. 2019).


Development of Orthogonal Fluorogenic Aptamers

RNA Mango aptamers can bind specifically to the green fluorogenic ligand TO1-R. RNA Peach preferentially binds TO3-R, a red fluorophore. These orthogonal aptamers demonstrate two-color fluorescence. (Kong et al. 2021)

RNA Mango aptamers can bind specifically to the green fluorogenic ligand TO1-R. RNA Peach preferentially binds TO3-R, a red fluorophore. These orthogonal aptamers demonstrate two-color fluorescence. (Kong et al. 2021)

Fluorogenic RNA aptamers are gaining interest as an alternative to fluorescent protein-based RNA tagging. In order to identify an aptamer-ligand pair that is orthogonal to our green RNA Mango and TO1-B system, we have performed a competitive in vitro selection to identify an RNA Peach aptamer (Kong et al. 2021). RNA Peach demonstrates a binding preference to the red TO3-B fluorophore, despite being then nearly identical chemical structure of TO3-B and TO1-B. Together, the RNA Mango and Peach pair demonstrate both binding and fluorescent orthogonality.  I am now working to further increase the orthogonality of fluorogenic aptamers systems.



 

Application of RNA Aptamers

Isothermal Amplification

Isothermal Amplification of SARS-CoV-2 with Mango NASBA

Isothermal nucleic acid amplification techniques can amplify nucleic acids without the need of a thermocycler. Coupled with our RNA Mango aptamer system, we have developed a more cost-effective and accessible system that produces its own fluorescent signal by transcription. Through careful primer design, our patented Mango NASBA technology is easily adapted to new targets and offers a tool for future point of care diagnostics (Abdolahzadeh et al. 2019). While RT-PCR tests can produce results with high specificity and sensitivity, isothermal fluorescent detection methods offer routes to cheaper and more robust testing.



FRET-based detection of RNA-RNA interactions and RNA-Protein interactions

 Secondary structure of the NiCo riboswitch and aptamer insertions (left). Relative organization of NiCo apta-FRET chimera sites upon folding (right) (Jeng et al. 2020).

 Secondary structure of the NiCo riboswitch and aptamer insertions (left). Relative organization of NiCo apta-FRET chimera sites upon folding (right) (Jeng et al. 2020).

To further understand the role of biological RNAs and their interactions with other biomolecules, new tools capable of measuring the folding, interactions and localization of RNA are needed. We utilize FRET which is an orientation and distance-dependent phenomena, to study such RNA interactions. The fluorescent RNA aptamers Broccoli and Mango-III have been used to measure the angular dependence of FRET, and this opens the way to improved structural interpretation of ensemble and single-molecule FRET measurements of RNA structures and their dynamics such as in the NiCo Riboswitch (Jeng et al. 2020).