Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
Taylor D. Canady,
April S. Berlyoung,
Joe A. Martinez,
Cole Emanuelson,
Cheryl A. Telmer,
Marcel P. Bruchez,
Bruce A. Armitage
Affiliations
Taylor D. Canady
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
April S. Berlyoung
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
Joe A. Martinez
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
Cole Emanuelson
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
Cheryl A. Telmer
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
Marcel P. Bruchez
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
Bruce A. Armitage
Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA
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.
