Targeting structured RNAs using cross-chiral recognition.
RNA is now widely recognized to modulate a variety of cellular outcomes beyond serving as a template for protein expression. As with other biomolecules, the function of RNA is closely related to its three-dimensional structure. Both secondary and tertiary structural motifs serve as important recognition elements for RNA-RNA and RNA-protein interactions. In addition, structured RNA elements play critical roles in a variety of diseases, including viral infections, cancer, and neurological disorders. Despite having a well-recognized therapeutic and diagnostic potential, structured RNAs remain underutilized targets for disease intervention (outside of antibiotics targeting the ribosome). Therefore, the development of new strategies for targeting structured RNAs is of the utmost importance.
The goal of this research project is to develop L-RNA-based affinity reagents and catalysts for practical biomedical applications. Our approach utilizes “cross-chiral” recognition, which we define as the hybridization independent recognition that occurs between two nucleic acids of opposite stereochemistry. Using in vitro selection techniques, we have prepared cross-chiral aptamers comprised of L-RNA (the nuclease resistant enantiomer of natural D-RNA) that bind structured D-RNA targets based on their unique shape rather than primary sequence. Having demonstrated the therapeutic potential of cross-chiral aptamers, we are now focused on improving their properties using a variety of approaches, including the use of unnatural nucleotides. In addition, we are using both biochemical and structural approaches to investigating how nucleic acids of opposite stereochemistry interact. Finally, we are utilizing cross-chiral recognition to develop biosensors capable of detecting dynamic RNA modification in real-time.
Use of chemical biology approaches to investigate relationships between chromatin structure and DNA modifications.
In recent years, chemically defined nucleosome arrays (often referred to as "designer" chromatin) have gained an increasingly important role in elucidating fundamental molecular mechanisms of chromatin regulation by enabling researchers to carry out quantitative measurements under precisely defined experimental conditions. Although numerous biochemical strategies have been reported for incorporating specific patterns of histone posttranslational modifications into designer chromatin, analogous methods for incorporating precisely positioned DNA modifications into these systems are lacking. This deficit precludes detailed molecular investigations of many important biological processes, including DNA repair and (de)methylation.
With this in mind, the Sczepanski lab recently developed a straightforward and versatile approach for incorporating precisely positioned DNA modifications into designer chromatin substrates. This approach allows precise control over the environment of a particular DNA modification, enabling a detailed analysis of the activities of associated protein factors in a variety of prescribed chromatin architectures. Emphasis is being placed on the process of DNA repair, and specifically base excision repair, as well as DNA (de)methylation. Our goal is to provide a much deeper understanding of the functional relationships between chromatin structure and DNA modifications, thereby facilitating the discovery of novel targets for disease intervention.
Engineering L-DNA nanodevices and circuitry capable of analyzing and manipulating molecular information in living systems.
DNA Nanotechnology is a rapidly growing area of research that encompasses topics ranging from nanoscale materials engineering to the development of sophisticated integrated synthetic biological circuitry. Not surprisingly, virtually all DNA nanotechnologies developed thus far are comprised of the native D-DNA stereoisomer. This is despite L-DNA, the synthetic enantiomer of native D-DNA, having numerous beneficial properties, including resistance to both nuclease degradation and off-target interactions with cellular components. Despite its many advantages, however, L-DNA is incapable of forming contiguous Watson-Crick base pairs with native D-nucleic acids. Consequently, the utility of L-DNA-based nanotechnology was previously considered to be extremely limited because it cannot be interfaced with endogenous D-nucleic acids, which serve as targets for the vast majority of biomedical applications.
Recently, the Sczepanski lab reported a novel methodology for interfacing L-DNA with native D-DNA using an oligonucleotide intermediary that is capable of hybridizing to both. This discovery enables, for the first time, development of DNA nanotechnology having fully-interfaced D- and L-oligonucleotide components. Moving forward, we are now interested in characterizing these novel reactions, designing more sophisticated reaction cascades, and inventing new and interesting DNA technologies that were not previously feasible in a homochiral context. We also seek to exploit this technology in order to engineer L-DNA nanodevices capable of analyzing and manipulating molecular information in living systems for the purpose of developing L-DNA based diagnostic and therapeutic devices. Beyond nucleic acid-integrated devices, we are also looking to expand on this technology by developing novel sensors for histone post-translational modifications based on heterochiral DNA logic circuits.