Refine
Has Fulltext
- yes (2)
Is part of the Bibliography
- yes (2) (remove)
Year of publication
- 2020 (2) (remove)
Document Type
- Doctoral Thesis (2)
Language
- English (2)
Keywords
- FRET (2) (remove)
Institute
Fluorogenic Aptamers and Fluorescent Nucleoside Analogs as Probes for RNA Structure and Function
(2020)
RNA plays a key role in numerous cellular processes beyond the central dogma of molecular biology. Observing and understanding this wealth of functions, discovering new ones and engineering them into purpose-built tools requires a sensitive means of observation. Over the past decade, fluorogenic aptamers have emerged to fill this niche. These short oligonucleotides are generated by in vitro selection to specifically interact with small organic fluorophores and can be utilized as genetically encoded tags for RNAs of interest.
The most versatile class of fluorogenic aptamers is based on derivatives of hydroxybenzylidene imidazolone (HBI), a conditional fluorophore mimicking the chromophore structure found in green and red fluorescent proteins. The respective aptamers are well-known by the “vegetable” nomenclature, including Spinach, Broccoli and Corn, and have found numerous applications for studying RNA function in vitro and in cells.
Their success, however, is somewhat overshadowed by individual shortcomings such as a propensity for misfolding, dependence on unphysiologically high concentrations of magnesium ions or, in the case of Corn, dimerization that might affect the function of the tagged RNA. Moreover, most fluorogenic aptamers exhibit limited ligand promiscuity by design, thereby restricting their potential for spectral tuning to a narrow window of wavelengths.
This thesis details the characterization of a new fluorogenic aptamer system nicknamed Chili. Chili is derived from an aptamer that was originally selected to bind 4-hydroxy-3,5-dimethoxy¬hydroxy-benzylidene imidazolone (DMHBI), resulting in a green fluorescent complex. Unlike other aptamers of its kind, Chili engages in a proton transfer cycle with the bound ligand, resulting in a remarkably large Stokes shift of more than 130 nm.
By means of an empirical ligand optimization approach, several new DMHBI derivatives were found that bind to Chili with high affinity, furnishing complexes up to 7.5 times brighter compared to the parent ligand. In addition, Chili binds to π-extended DMHBI derivatives that confer fluorescence in the yellow–red region of the visible spectrum. The highest affinity and degree of fluorescence turn-on for both green and red fluorogenic ligands were achieved by the incorporation of a unique, positively charged substituent into the HBI scaffold.
Supplemented by NMR spectroscopy, kinetic and thermodynamic studies showed that the binding site of Chili is loosely preorganized in the absence of ligand and likely forms a G-quadruplex upon ligand binding.
To showcase future applications, Chili was incorporated into a FRET sensor for monitoring the cleavage of an RNA substrate by a 10-23 DNAzyme.
Besides aptamers as macromolecular fluorescent complexes, fluorescent nucleobase analogs are powerful small isomorphic components of RNA suitable for studying structure and folding. Here, the highly emissive nucleobase analog 4-cyanoindole (4CI) was developed into a ribonucleoside (r4CI) for this purpose. A new phosphoramidite building block was synthesized to enable site-specific incorporation of 4CI into RNA.
Thermal denaturation experiments confirmed that 4CI behaves as a universal nucleobase, i.e. without bias towards any particular hybridization partner. Photophysical characterization established r4CI as a generally useful fluorescent ribonucleoside analog. In this work, it was employed to gain further insight into the structure of the Chili aptamer. Using several 4CI-modified Chili–HBI complexes, a novel base–ligand FRET assay was established to obtain a set of combined distance and orientation restraints for the tertiary structure of the aptamer.
In addition to their utility for interrogating structure and binding, supramolecular FRET pairs comprising a fluorescent nucleobase analog donor and an innately fluorogenic acceptor hold great promise for the construction of color-switchable RNA aptamer sensor devices.
Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor.