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.
Highlights
IntroductionHigh affinity recognition and accurate discrimination of nucleic acids is the foundation for many biotechnology tools and biomedical applications ranging from PCR-based diagnostics and fluorescence in situ hybridization (FISH) to antisense blocking of translation and genome editing
High affinity recognition and accurate discrimination of nucleic acids is the foundation for many biotechnology tools and biomedical applications ranging from PCR-based diagnostics and fluorescence in situ hybridization (FISH) to antisense blocking of translation and genome editing.the enduring challenge of technologies that rely on nucleic acid hybridization is the trade-off between affinity and selectivity [1]
To test the impact of secondary structure on γPNA hybridization selectivity, we designed in other contexts where the target site is more accessible is susceptible to off-target struc_γPNA, which consists of a 10 nucleotide target-recognition domain composed of γPNA
Summary
High affinity recognition and accurate discrimination of nucleic acids is the foundation for many biotechnology tools and biomedical applications ranging from PCR-based diagnostics and fluorescence in situ hybridization (FISH) to antisense blocking of translation and genome editing. The enduring challenge of technologies that rely on nucleic acid hybridization is the trade-off between affinity and selectivity [1]. On the other hand, optimizing the selectivity of a probe for its target is much more difficult due to the demand for discriminating single mismatches. Making the probe short so that a mismatch prevents hybridization at the experimental temperature raises the likelihood that other perfectly matched sequences will be present elsewhere in the genome/transcriptome, leading to off-target hybridization. Making the probe long in order to find a unique sequence enables off-target hybridization due to the modest destabilizing effect of a single mismatch in a high-affinity probe
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