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Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

ABSTRACT: High affinity nucleic acid analogues such as gammaPNA (?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 ?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 ?PNA probe, leaving an additional five ?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.

SUBMITTER: Canady TD

PROVIDER: S-EPMC7070858 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

Canady Taylor D TD Berlyoung April S AS Martinez Joe A JA Emanuelson Cole C Telmer Cheryl A CA Bruchez Marcel P MP Armitage Bruce A BA

Molecules (Basel, Switzerland) 20200221 4

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 ...[more]

PMID: 32098111

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Project description:In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.

Dataset Information

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

Publications

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

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