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The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman’s rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.

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).

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 along with challenging experimental conditions requirements (homopurine targets; denaturing steps; low ionic strengths; short target sequences). The 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 for specific detection of chromosomal DNA in 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 labelled isolated nuclei, offering a potential advantage over the laborious microscope evaluation of detection (CHAPTER 3).

Outbreaks in the US of highly pathogenic avian influenza virus (H5N1) in dairy cows have been occurring for months creating new possibilities for direct contact between the virus and humans. Eisfeld examined the pathogenicity and transmissibility of a bovine HPAI H5N1 virus isolated from New Mexico in a series of and assays. They found the virus has a dual human- and avian virus-like receptor-binding specificity as measured in a solid phase glycan binding assay. Here, we examined the receptor specificity of a bovine HPAI H5N1 virus (A/bovine/OH/B24OSU-432/2024, H5N1, clade 2.3.4.4b) employing four different assays including glycan array technology, bio-layer interferometry (BLI), a solid phase capture assay and hemagglutination of glycan remodeled erythrocytes. As controls, well characterized avian (A/Vietnam/1203/2004, H5N1, clade 1) and human (A/CA/04/2009, H1N1) IAVs were included that bind α2,3- and α2,6-sialosides, respectively. We found that A/bovine/OH/B24OSU-432/2024 preferentially binds to \"avian type\" receptors (α2,3-sialosides). Furthermore, sequence alignments showed that A/bovine has maintained amino acids in its HA associated with α2,3-sialoside (avian) receptor specificity. We conclude that while we find no evidence that A/bovine has acquired human virus receptor binding specificity, ongoing efforts must be placed on monitoring for this trait.