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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...

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Autores principales: Canady, Taylor D., Berlyoung, April S., Martinez, Joe A., Emanuelson, Cole, Telmer, Cheryl A., Bruchez, Marcel P., Armitage, Bruce A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070858/
https://www.ncbi.nlm.nih.gov/pubmed/32098111
http://dx.doi.org/10.3390/molecules25040970
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author Canady, Taylor D.
Berlyoung, April S.
Martinez, Joe A.
Emanuelson, Cole
Telmer, Cheryl A.
Bruchez, Marcel P.
Armitage, Bruce A.
author_facet Canady, Taylor D.
Berlyoung, April S.
Martinez, Joe A.
Emanuelson, Cole
Telmer, Cheryl A.
Bruchez, Marcel P.
Armitage, Bruce A.
author_sort Canady, Taylor D.
collection PubMed
description 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.
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spelling pubmed-70708582020-03-19 Enhanced Hybridization Selectivity Using Structured GammaPNA Probes Canady, Taylor D. Berlyoung, April S. Martinez, Joe A. Emanuelson, Cole Telmer, Cheryl A. Bruchez, Marcel P. Armitage, Bruce A. Molecules Article 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. MDPI 2020-02-21 /pmc/articles/PMC7070858/ /pubmed/32098111 http://dx.doi.org/10.3390/molecules25040970 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Canady, Taylor D.
Berlyoung, April S.
Martinez, Joe A.
Emanuelson, Cole
Telmer, Cheryl A.
Bruchez, Marcel P.
Armitage, Bruce A.
Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title_full Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title_fullStr Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title_full_unstemmed Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title_short Enhanced Hybridization Selectivity Using Structured GammaPNA Probes
title_sort enhanced hybridization selectivity using structured gammapna probes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070858/
https://www.ncbi.nlm.nih.gov/pubmed/32098111
http://dx.doi.org/10.3390/molecules25040970
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