Harnessing XNAs to Expand the Genetic Toolkit for RNA Silencing and RNA editing

About This Grant

PROJECT SUMMARY The objective of my research program over the next five years is to advance nucleic acid chemistry and harness in vitro selection (SELEX) techniques to expand the genetic toolkit for RNA silencing and editing, laying a foundation for next-generation RNA therapeutics. RNA silencing and editing hold transformative potential for treating genetic disorders caused by single-point mutations without altering the genetic code. However, current RNA silencers lack sufficient allele specificity, and site-directed RNA adenosine-to-inosine (A-to-I) editing remains in its early stage. To address these challenges, my program focuses on the chemistry, structure, and function of de novo XNAs (xeno nucleic acids)—synthetic nucleic acid analogs with modified backbones that enhance nuclease resistance, extend half-life, and confer unique thermodynamic properties. While chemical backbone modifications have revolutionized RNA therapeutics, they often compromise the natural functionality of catalytic RNAs or aptamers, when incorporated after the evolution process. To overcome these limitations, we are developing an innovative XNA-SELEX platform to generate functional XNA molecules. Over the past five years, I have extensively investigated the effects of chemical modifications on nucleobases and sugar backbones, analyzing their impact on RNA base pairing, self-assembly, and structure. Alongside, I have developed enzymatic and non-enzymatic tools for RNA/XNA manipulation. These efforts form a strong groundwork for creating functional XNAs to address biomedical challenges. Building on this work, we aim to design novel XNAzymes and XNA aptamers based on 3′-NP-DNA (N3′-P5′ linked phosphoramidate DNA) and 2′-F-RNA (2′-fluoro-RNA). These XNAs exhibit enhanced thermal stability, rigid secondary structures, enzymatic resistance, and biocompatibility, making them ideal for allele-specific silencing and precise recruitment of protein enzymes for RNA editing. Their performance will be optimized in cellular models to achieve efficient silencing and editing of disease-associated alleles. In parallel, we will investigate the tertiary structures and folding properties of 3′-NP-DNA and 2′-F-RNA to uncover novel structural motifs and elucidate their functional roles. These findings will guide the design of next-generation XNAzymes and aptamers, expanding their therapeutic potential for RNA targeting and editing. By integrating chemical biology, structural analysis, and functional validation, this program aims to establish a robust platform for XNA-based technologies. The compact size and enhanced stability of XNAzymes and aptamers provide significant advantages for in vivo delivery, addressing key barriers in therapeutic RNA silencing and editing. This work will advance both fundamental insights and translational applications, paving the way for innovative XNA technologies in biotechnology and medicine.