| dc.description.abstract | Nature is inherently homochiral. L-DNA and L-RNA, the enantiomeric forms of native D-DNA and D-RNA respectively, do not occur naturally and are virtually bioorthogonal. Compared to common chemical modifications, L-DNA/RNA exhibit superior qualities such as increased biostability due to exceptional nuclease resistance, low immunogenicity, and minimal off-target effects. When interacting with an achiral physical or chemical environment, they behave similar to their native D-counterparts, facilitating easy optimization of designs based on L-DNA/RNA. However, while these “mirror-image” nucleic acids hybridize to each other, they are incapable of forming contiguous WC base-pairs with complementary native nucleic acids, a caveat that until recently precluded their use in applications at the interface of native biology. Redirecting the focus toward non-canonical heterochiral nucleic acid interactions based on non-WC hydrogen bonding, Van der Waals and hydrophobic interactions etc., overcomes this obstacle. This work focuses on exploring and expanding these novel recognition modalities with the aim of interfacing L-DNA/RNA with native nucleic acids using both structure and sequence-based approaches.
Complex structural interactions in RNA govern almost all aspects of gene expression. Targeting structured RNA is thus integral to our fundamental understanding of RNA biology as well as in the development of therapeutics. Several heterochiral L-DNA/RNA aptamers have been evolved to bind their respective RNA targets with high specificity and affinity, in a structure-specific manner. In contrast, evolution of L-ribozymes is more challenging. RNA ligation and polymerization is the only reported example of heterochiral catalysis to date. In the first part of my thesis, I will discuss the in vitro evolution of a heterochiral ribonuclease ribozyme that interacts with a representative structured RNA target to mediate phosphodiester bond scission resulting in cleavage of the target. This opens a novel route to chemically target a specific RNA within its structural context while eliminating WC-based off-target hybridization. Furthermore, this approach is also promising for future therapeutic applications.
The second part of this thesis focuses on sequence-specific interfacing strategies. Our research group recently described a novel technique called “heterochiral” DNA strand displacement reactions that utilize an achiral peptide nucleic acid (PNA) mediator to exchange sequence information between D-DNA and L-DNA, thus providing a route to exploit the advantageous properties of L-DNA in dynamic DNA nanotechnology applications. In this work I present extensive kinetic characterization of these novel reactions by systematically varying key design parameters in order to establish a set of design principles that will facilitate the rational design of such devices for biomedical applications in the future. Additionally, investigation of the biophysical mechanism of these reactions reveals a novel stereochemical control over reaction kinetics, that adds to the versatility of future designs. | |
