Abstract
Interest in developing probes capable of targeting chromosomal DNA in cells has grown to meet diagnostic and therapeutic needs. DNA has a stable predicable double-stranded structure that has been the subject of study to identify agents that can specifically bind to the duplex (CHAPTER 1). Success stories from probe technologies such as triplex-forming oligonucleotides, peptide nucleic acids (PNAs), and minor-groove-binding polyamides have been well characterized. However, they suffer limits of detection with experimental conditions requirements (homopurine targets; denaturing steps; low ionic strengths; short target sequences). Newly discovered CRISPR-Cas9 has garnered much attention; however, the approach requires transfection of plasmids encoding CRISPR-Cas9 components. To address these shortcomings, our laboratory has developed Invader probes. Placement of 2'-O-(pyren-1-yl)methyl RNA monomers in +1 interstrand zipper arrangements destabilizes the probe duplex as the intercalating pyrene moieties vie for the same space between two Watson-Crick base pairs. These ‘energetic hotspots’ activate the double-stranded probe and, in concert with the high affinity for complementary DNA (cDNA) displayed by individual probe strands, provide the driving force for recognition of mixed-sequence target sites. The capabilities and features of Invader probes specific detection of chromosomal DNA fluorescent in situ hybridization (FISH) assays at near physiological conditions were statistically analyzed. Based on these results, Optimized Invader probes were synthesized and showed improved efficiency in chromosomal DNA detection (CHAPTER 2). Moreover, the combination of Invader FISH probes with the powerful detection properties of flow cytometry was used to quantify thousands of specifically label isolated nuclei, offering a potential advantage over the laborious microscope evaluation of detection (APPENDIX A).