Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Understanding the Two-Dimensional Mixing Behavior of 1-Naphthalenethiol and Octanethiol.

A two-dimensional (2D) mixture in the form of a self-assembled monolayer composed of two distinct organothiol compounds was created by sequentially depositing 1-naphthalenethiol (1NT) and octanethiol (OT) on a gold surface. By varying the sequence of deposition, two mixed surface systems were created. The surface structure of the resulting mixed monolayer was characterized with Scanning Tunneling Microscopy (STM) and showed surface disorder across all investigated domains. Elemental analysis was carried out with X-ray Photoelectron Spectroscopy (XPS) and indicated that the 1NT monolayer was prone to significant oxidation. Reductive desorption (RD) was used to characterize the binding strength and electrochemical environments of the molecular components in the mixture, and confirmed disordered molecular layers. Due to the presence of oxidized species in the 1NT monolayer, 1NT was displaced by OT resulting in a novel surface structure composed of either OT or 1NT. Monolayers of OT that were exposed to a solution of 1NT resulted in disordered surface structures with a significant amount of gold vacancy islands. To date, there is no experimental phase diagram explaining the chemical behavior of two-dimensional mixtures. This study addresses the need for an experimental understanding of the phase behavior of mixed organothiol self-assembled monolayers (SAMs).