Enhanced Hybridization Selectivity Using Structured GammaPNA Probes

Abstract

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.

Peptide Nucleic Acid with Double Face: Homothymine–Homocytosine Bimodal Cα-PNA (bm-Cα-PNA) Forms a Double Duplex of the bm-PNA2:DNA Triplex

“…Complexes of PNA with DNA/RNA have specificity and affinity higher than that of analogous DNA:DNA/DNA:RNA complexes . They have potential utility in nucleic acid therapeutics and proven applications as molecular probes in diagnostics . Their limitations in terms of solubility and poor cell permeability have been addressed widely through various chemical modifications .…”

Structural Design and Synthesis of Bimodal PNA That Simultaneously Binds Two Complementary DNAs To Form Fused Double Duplexes

“…Based on choice of sequences (polypurines/polypyrimidines/self-complementary) on either side, bimodal PNAs can lead to fused duplexes, triplexes, and tetraplexes (G n /C n ) with cDNA. They can be engineered to yield supramolecular functional nanostructures for probable applications in PNA material science . Distant biotechnological applications include simultaneous targeting of two genes or micro RNA structures for therapeutic purposes, and, as equivalents of short DNA staples, they can be active in programmed folding of plasmids in DNA origami …”

Cγ(S/R)-Bimodal Peptide Nucleic Acids (Cγ-bm-PNA) Form Coupled Double Duplexes by Synchronous Binding to Two Complementary DNA Strands

“…Based on the choice of the sequences on either side (polypurines/polypyrimidines/G n /C n ), the Cγ- and Cα-bimodal PNAs can be used to generate welded PNA:PNA binary duplexes, ternary double triplexes, and tetraplexes. The enhanced stability and augmented molecular recognition properties of the bimodal PNAs can be exploited to design complex supramolecular nanoassemblies with defined functions tailored for various applications in biotechnology as well as material science. , Among the probable biological applications, bimodal PNAs can be used to specifically and concurrently target two genes, inhibit micro RNA structures, to probe biological processes and modulate them for therapeutics . Further, bimodal PNAs can be used like stapler strands in DNA origami to expand the limits of programmable folding in nucleic acid nanotechnology …”