Advancements in RNA 2'-hydroxyl covalent modification : novel technologies and chemical insights

The unusual nucleophilicity of 2′-OH groups on RNA molecules has proven broadly useful for investigating and conjugating this biomolecule. Carefully designed electrophilic small molecules can selectively modify RNA 2′-OH, creating adducts that may often be difficult to generate using other techniques like solid-phase oligonucleotide synthesis or enzymatic RNA synthesis. By targeting 2′-OH groups instead of nucleobase structures, this modification strategy is sequence-independent and RNA-selective (over DNA) by design. The reagents employed in this approach are also generally sensitive to 2′-OH environment in RNA: preferentially reacting at non-base paired nucleotides over base-paired locations. The importance of this technology is demonstrated by salient applications in the investigation of RNA secondary structure inside live cells, control of RNA function, and enhancement of messenger RNA stability, among many others. Although highly useful, until recently RNA 2′-OH chemical modification technology has had several limitations. The small molecule electrophiles employed in this approach have classically been highly reactive acylating reagents, with short half-lives in water limiting their utility particularly in cellular studies. These reagents are also generally difficult to purify and store, and require >10% DMSO composition (by volume) in reactions for preparative levels of RNA conversion. The possibility of generating novel covalent linkages at RNA 2′-OH beyond just the ester and carbonate moieties created by these reagents has also never been explored. In addition, strategies to direct 2′-OH acylation towards base-paired regions of RNA, or to achieve site-selective RNA modification using a catalytic approach, have not yet been developed. Finally, the effect of chirality in small molecule acylating agents have also never been investigated. This work herein presents the development of novel technologies for 2′-OH-selective RNA chemical modification to overcome many of the current limitations, and reports discoveries of new chemical insights that may broadly inform future work on RNA chemical modification. Chapter 1 describes the synthesis and characterization of a series of acylating reagents possessing fluorescence properties. The design of these molecules aims to target and selectively modify base-paired regions of RNA through intercalative scaffolds and/or electrostatic interactions. It presents the optimization of RNA reaction conditions to enhance 2′-OH acylation yields and investigates the potential of using these molecules as innovative reporters of RNA secondary structure through reverse transcription-stop (RT-stop) analysis. The ability of these reagents to demonstrate a fluorescent light-up signal in presence of RNA is also tested. The data and results summarized in this chapter demonstrate that for the reagents tested, the presence of nucleic acid-binding scaffolds and fluorogenic motifs do not lead to selective reactivity at base-paired regions or a significant fluorescence light-up upon RNA modification. Chapter 2 presents the synthesis and characterization of a series of DNA oligonucleotides modified with nucleophilic transfer catalysts. The design of these modified oligonucleotides aims to achieve covalent modification at a specific 2′-OH in an RNA sequence, by utilizing sequence complementarity to the target RNA, without the need for superstoichiometric quantities of complementary DNA by leveraging nucleophilic catalysis. This chapter presents the exploration of RNA reaction conditions with a variety of RNA 2′-OH modifying reagents, and characterization of modification by using RT-stop analysis (via PAGE gel electrophoresis). The early data obtained for this study, presented in this chapter, indicate that all the tested combinations of catalyst-containing oligonucleotides and RNA modifying small molecule reagents do not achieve site-selective modification in a test substrate single-stranded RNA. Chapter 3 describes RNA Sulfonylation, a novel chemical technology for 2′-OH selective RNA covalent modification. It reports that many activated small-molecule sulfonyl species can exhibit extended lifetimes in water and retain 2′-OH reactivity. The data establish favorable aqueous solubility for selected reagents and successful RNA-selective reactions at stoichiometric and superstoichiometric yields, particularly for aryl sulfonyltriazole species. Sulfonyltriazoles are more stable than most prior carbon electrophiles by orders of magnitude in aqueous environments, and tolerate silica chromatography. Furthermore, an azide-substituted sulfonyltriazole reagent is described to introduce labels into RNA via click chemistry. Like acylation, sulfonylation occurs with selectivity for unpaired nucleotides over those in the duplex structure, and a sulfonate adduct causes reverse transcriptase stops, suggesting future use in RNA structure analysis. Probing of rRNA is demonstrated in human cells, indicating possible cell permeability. Chapter 4 outlines the investigation of a series of chiral acylimidazole reagents with varying steric hindrance near the electrophilic center. It describes experiments that aim to determine yields and diastereoselectivity in RNA reaction for the tested compounds. Timecourse analysis of RNA acylation for a subset of enantiomeric reagent pairs reveals new insights into preferred structural features for high reactivity and diastereoselectivity. This study establishes the importance of chirality in small molecule RNA modifying reagents, with insights that may inform future design of RNA targeting molecules. Chapter 5 introduces RNA Arylation, another novel chemical technology for 2′-OH selective RNA covalent modification. It presents the investigation of the RNA reactivity of a series of electrophilic aromatic reagents with varying structural scaffolds. It describes screening experiments that aim to determine yields and 2′-OH selectivity in RNA reaction for the tested compounds. Importantly, many of these compounds are highly water-soluble, and for the first time RNA modification reactions with >99% yields are reported in total absence of any organic co-solvent which may be highly useful for future biological applications. Many of the reported molecules are also highly stable in water, with half-lives of days, while possessing a surprising ability to modify RNA with preparative yields within hours. The facile synthesis, purification, and applications in RNA conjugation using Cu-free click chemistry are described. This study introduces RNA 2′-OH Arylation as a promising technology that overcomes the limitations of prior RNA modification techniques.

Keywords: Humanities and Sciences.
Chemistry.
Advisor: Kool Eric T.
Cegelski Lynette
Waymouth Robert M.