Abstract
Molecular probes that are capable of sequence-specific recognition of double-stranded DNA (dsDNA) in physiologically relevant conditions have tremendous potential as tools in diagnostics and therapeutics against genetic diseases. Pioneering strategies towards dsDNA-recognition included minor groove binding pyrrole-imidazole polyamides, and major groove binding triplex forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs). These approaches are effective at recognizing nucleobase-specific features from the duplex grooves, although with significant limitations; polyamides are restricted to very short sequences due to binding and sequence complementarity, while TFOs and PNAs can only target polypurine regions. Advancements in affinity-enhancing nucleic acid-mimics have enabled alternative DNA-binding modes; namely, strand-invasion mechanisms. These probes invade the existing Watson-Crick base pairs of DNA to form new, more stable Watson-Crick base pairs between probe and target strands. Our lab has introduced an effective strand-invading strategy: Invader probes, i.e., short DNA duplexes modified with one or more +1 interstrand zipper arrangements of intercalator-modified nucleotides. In Chapter 1 of this Dissertation, I will discuss the development and applications of Invader probes since the synthesis of the very first Invader building block twenty years ago. In more recent years, we have focused on developing alternative Invader probe architectures in effort to improve probe lability, specificity, and dsDNA-affinity. My contributions to these investigations have involved the development of chimeric probe duplexes consisting of individual Invader strands and complementary Xeno RNAs (XRNAs) like locked nucleic acid (LNA), 2'-O-methoxyethyl (MOE), 2'-O-methyl (O2'-Me), and 2'-Fluoro (2'-F). Thus, in Chapter 2, I present my work investigating chimeric Invader:LNA probes in our model mixed-sequence context for which I evaluated this probe architecture with varying modification density of the Invader strand. In Chapter 3, I further explore chimeric Invader:LNA probes in pathogen-specific sequence contexts. Additionally in Chapter 3, I report a comprehensive protocol for evaluating DNA-targeting oligonucleotide-based probes via electrophoretic mobility shift assays. Lastly, in Chapter 4, I report on the biophysical and dsDNA-recognition properties of chimeric Invader:XRNA probes with XRNAs being fully modified with MOE, O2'-Me, 2'-F or LNA/MOE mixmers. Indeed, these investigations have led to the discovery of highly efficient chimeric Invader:XRNA probe duplexes and single-stranded LNA/MOE mixmers, with some probe designs achieving near-stoichiometric recognition of dsDNA with single base pair accuracy. The distinctive properties of these probes unlock exciting possibilities in molecular biology and constitute valuable additions to the molecular toolbox for DNA-targeting applications.