Chemical modifications for spectral tuning of fluorogenic RNA aptamers

Fluorogenic RNA aptamers such as Spinach, Corn, and Mango have provided a practical method for imaging RNAs. While much work has gone into developing different aptamer-ligand systems to expand the current toolbox of fluorogenic RNA aptamers, the potential of synthetic modifications has remained largely unexplored. To address this gap, I have investigated a range of canonical mutations, naturally occurring noncanonical modifications, and artificial nucleotide labels to study the effects of these synthetic modifications on the spectral properties of fluorogenic RNA aptamers. To access naturally occurring, noncanonical RNA modifications, I have synthesized novel phosphoramidites for tRNA modifications PreQ0 (2-amino-5-cyano-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidin-4-one) and wyosine (3,4-dihydro-4,6-dimethyl-3-β-D-ribofuranosyl-9H-imidazo[1,2-a]purin-9-one) for incorporation into an RNA aptamer using solid-phase oligonucleotide synthesis. Guided by the crystal structure of the Spinach aptamer, I developed a Split Baby Spinach aptamer which is accessible via solid-phase oligonucleotide synthesis. I then explored the effects of modifications such as PreQ0 and wyosine, as well as fluorescent nucleotide analogues 2-aminopurine and pyrrolocytosine, on the fluorogenic aptamer when bound to different fluorophore-ligands. While most mutations had a negligible effect on the fluorescent properties of the aptamer-fluorophore complex, one wyosine mutation near the ligand binding pocket exhibited enhanced fluorescence turn-on. Finally, guided by the crystal structure of iMango-III, I have also individually introduced the fluorescent dyes, fluorescein and rhodamine, into five positions surrounding the iMango-III ligand binding site. These modifications create an internal FRET system, providing a simple method for tuning the excitation or emissions wavelength of the RNA aptamer to those of the introduced fluorophores. These synthetic modifications demonstrate the potential of noncanonical nucleotides for fluorescence modulation enhancement.