γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats

Secondary Structure Analysis of Intra- and Intermolecular Guanine Quadruplexes and Targeting through Complementary and Homologous Binding using Peptide Nucleic Acid

Many of the projects discussed in this thesis utilize one or more of the binding modes shown in Figure 1.12. As a result of this work, we expand our understanding of GQ secondary structures and show the ability to target these GQs using multiple binding modes. Chapter 2 analyzes variations on a short peptide nucleic acid (PNA) probe that homologously binds to guanine-rich telomeric DNA. We compare the PNA probe with and without backbone modifications and utilize a GQ binding small molecule fluorophore; a combination of two binding modes. This work identifies that TO-labeled probes show significantly increased thermal stability compared to their unlabeled counterparts. Modifications inducing a right handed helix in the PNA show no difference in thermal stability. This short probe does not possess selectivity as evidenced by in vitro translation experiments performed in cell lysate. In Chapter 3, we expand the current literature on RNA:DNA heteroquadruplexes (RDQ). This GQ contains both RNA and DNA guanine tracts and has recently been identified as a regulator between transcription and translation. We show the formation and stabilities of three suspected RDQ sequences using an optimized scaffold duplex. The formation of the RDQs was also monitored with a small molecule fluorophore, Thioflavin T.Chapter 4 utilizes the RDQ systems created in Chapter 3 and compares them to DNA and RNA homoquadruplexes. With this chapter, we make DNA:DNA and RNA:RNA homoquadruplexes using the scaffold duplex created in chapter 3 and compare their biophysical properties to our RDQs. A modified PNA oligomer is then used to duplex invade each heteroquadruplex. We identify that RDQs possess characteristics of both RNA and DNA heteroquadruplexes. Our PNA probe is also able to sufficiently target each heteroquadruplex with varying IC50 values. Chapter 5 describes the use of dual gammaPNA (γPNA) probes that hybridize to alternating sites along a telomere. Each probe contains a fluorophore, and can undergo Förster resonance energy transfer (FRET). We study the biophysical characteristics of these probes and observe bright staining of telomeres in nuclei. Finally, we show the incorporation of additional fluorophores does not significantly improve FRET efficiency. <br

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes

High affinity nucleic acid analogues such as gammaPNA (&gamma;PNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a &gamma;PNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the &gamma;PNA probe, leaving an additional five &gamma;PNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity