Fluorescent nucleoside analogues to advance RNA-centered therapeutics

Abstract This research program seeks to create synthetically modified, fluorescent nucleosides and oligonucleotides and apply them to (i) imaging the uptake and endosomal escape of therapeutic oligonucleotides and (ii) identifying ligands that bind specifically to RNA targets and modulate biology. Nucleic acid-centered medicine is important because it presents a new opportunity to treat diseases that have been challenging in traditional medicinal chemistry. We seek to address three important problems in nucleic acids chemistry that will be enabling to RNA- centered medicine. (1) When nucleic acid-based medicines, including antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), and mRNA vaccines are delivered to cells, they are taken up in endosomes, from which < 5% of these drugs escape and are active. (2) Targeting RNA directly with small molecules has encountered problems with ligand promiscuity; there is a need for additional screening methods for selective ligand discovery. (3) Synthetic methodologies available for making C-ribosides, which are valuable in medicinal chemistry, chemical biology and to make fluorescent probes, are much harsher and more limited in scope than those for making C-2′-deoxyribosides. To address these challenges, we propose the following research plans. (1) We will create modified ASO and siRNA surrogates incorporating a pair of fluorescent ribonucleosides where one serves as an environmentally insensitive reference and the second as a pH-responsive probe that will differentiate endosomal from cytosolic pH. These modified ASOs and siRNAs will be used for live-cell imaging of RNA uptake and quantification of endosomal escape. (2) We will apply fluorescent nucleoside analogues to the creation of riboswitch and RNA hairpin constructs designed to detect ligand binding that is both specific and effective for modulating biology. These will be useful for studying ligand binding to therapeutically relevant RNA and for discovering new ligands in high-throughput screening. (3) We will develop new synthetic methods for making C-ribonucleosides that will have broad utility in chemical biology and medicinal chemistry. Together, these research directions aim to advance nucleic acids chemistry as applied to RNA-centered therapeutics.