Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide
Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation ba...
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doaj-0f5e3d8d27384b4280384e6903a127ac2020-11-25T03:34:14ZengMDPI AGCells2073-44092020-10-0192375237510.3390/cells9112375Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense OligoribonucleotideDorota Gudanis0Damian Kaniowski1Katarzyna Kulik2Daniel Baranowski3Zofia Gdaniec4Barbara Nawrot5Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, PolandCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Lodz, PolandCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Lodz, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, PolandCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Lodz, PolandAntisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA <i>levels</i> compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). <i>In</i> addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems.https://www.mdpi.com/2073-4409/9/11/2375G-quadruplexquadruplex-duplex hybridRNA G-rich antisense oligonucleotideEGFR mRNAselective G4 fluorescent probeoBMVC derivative |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Dorota Gudanis Damian Kaniowski Katarzyna Kulik Daniel Baranowski Zofia Gdaniec Barbara Nawrot |
spellingShingle |
Dorota Gudanis Damian Kaniowski Katarzyna Kulik Daniel Baranowski Zofia Gdaniec Barbara Nawrot Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide Cells G-quadruplex quadruplex-duplex hybrid RNA G-rich antisense oligonucleotide EGFR mRNA selective G4 fluorescent probe oBMVC derivative |
author_facet |
Dorota Gudanis Damian Kaniowski Katarzyna Kulik Daniel Baranowski Zofia Gdaniec Barbara Nawrot |
author_sort |
Dorota Gudanis |
title |
Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide |
title_short |
Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide |
title_full |
Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide |
title_fullStr |
Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide |
title_full_unstemmed |
Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells Between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide |
title_sort |
formation of an rna quadruplex-duplex hybrid in living cells between mrna of the epidermal growth factor receptor (egfr) and a g-rich antisense oligoribonucleotide |
publisher |
MDPI AG |
series |
Cells |
issn |
2073-4409 |
publishDate |
2020-10-01 |
description |
Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA <i>levels</i> compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). <i>In</i> addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems. |
topic |
G-quadruplex quadruplex-duplex hybrid RNA G-rich antisense oligonucleotide EGFR mRNA selective G4 fluorescent probe oBMVC derivative |
url |
https://www.mdpi.com/2073-4409/9/11/2375 |
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