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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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. |
format | Online Article Text |
id | pubmed-7070858 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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