Project Summary/Abstract The increasing appreciation of RNA’s structure-function relationships has led to a demand for new technologies that enable targeting of specific RNA structures. However, discovery of molecules that are capable of binding RNA structures with high affinity and selectivity has proven challenging using current approaches, resulting in a technological gap that precludes the development of new research tools to study RNA function and therapeutics to treat RNA-mediated diseases. Thus, development of new technologies that enable structure- specific targeting of RNA remains an important challenge in many fields. The central vision of my research program is to address the deficit of structure-specific RNA-binding reagents using a radically different type of nucleic acid affinity reagent: L-aptamers. L-Aptamers are unique because they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. Because oligonucleotides of opposite stereochemistry (D versus L) are incapable of forming contiguous Watson-Crick base pairs with each other, we are able to evolve L-aptamers that adaptively bind structured D-RNA targets through tertiary interactions (shape) rather than primary sequence. This unique “cross-chiral” mode of recognition occurs with high affinity and selectivity, and these interactions can modulate the function of RNA targets through several modes of action. These properties, coupled with the intrinsic nuclease resistance of L-oligonucleotides, provide L-aptamer technology a broad range of opportunities in biomedical research and disease intervention. During the next five years, my research group aims to further develop L-aptamer technology to realize its promise as a practical research and therapeutic tool. We aim to improve the RNA-binding properties of L- aptamers using new in vitro selection strategies and to improve our understanding of cross-chiral recognition by providing the first three-dimensional structure of an L-aptamer-RNA heterochiral complex. Building on our prior work, we will continue to develop L-aptamers as inhibitors of oncogenic microRNAs, representing a promising therapeutic strategy for related cancers. However, we will also pursue several novel applications of L-aptamer technology, including RNA-targeted small molecule drug discovery and intracellular RNA imaging, thereby substantially broadening the impact of this work. Finally, we aim to comprehensively characterize the behavior of L-oligonucleotides in living cells — how they interact with these environments and the potential consequences — the results of which will have a broad impact on the development of all future L-oligonucleotide-based biotechnologies, including L-aptamers.