Assembly and Characterization of RNA/DNA Hetero-G-Quadruplexes.

Transient association of guanine-rich RNA and DNA in the form of hetero-G-quadruplexes (RDQs) has emerged as an important mechanism for regulating genome transcription and replication but relatively little is known about the structure and biophysical properties of RDQs compared with DNA and RNA homo-G-quadruplexes. Herein, we report the assembly and characterization of three RDQs based on sequence motifs found in human telomeres and mitochondrial nucleic acids. Stable RDQs were assembled using a duplex scaffold, which prevented segregation of the DNA and RNA strands into separate homo-GQs. Each of the RDQs exhibited UV melting temperatures above 50 °C in 100 mM KCl and predominantly parallel morphologies, evidently driven by the RNA component. The fluorogenic dye thioflavin T binds to each RDQ with low micromolar KD values, similar to its binding to RNA and DNA homo-GQs. These results establish a method for assembling RDQs that should be amenable to screening compounds and libraries to identify selective RDQ-binding small molecules, oligonucleotides, and proteins.

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

High affinity nucleic acid analogues such as gammaPNA (γPNA) 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 γPNA 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 γPNA probe, leaving an additional five γPNA 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.

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.