Abstract
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 levels compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). In 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.
Highlights
G-quadruplexes (G4s) are DNA or RNA structures formed from stacked guanine tetrads.Computational studies have revealed the prevalence of more than 370,000 candidate putativeG-quadruplex DNA sequences (PQS) in various regions of the human genome [1]
To increase the propensity of target sequences containing two distant G-tracts to fold into a G-quadruplex after hybridization with Q-a so-called “guide” fragment (ASO), we obtained two model oligonucleotides bearing the chemical modifications known to stabilize DNA G-quadruplexes [34,35,36]
We propose an approach for downregulation of genes containing two distant G-tracts in a G4-mediated manner using RNA G-rich antisense oligonucleotides
Summary
G-quadruplexes (G4s) are DNA or RNA structures formed from stacked guanine tetrads.Computational studies have revealed the prevalence of more than 370,000 candidate putativeG-quadruplex DNA sequences (PQS) in various regions of the human genome [1]. G-quadruplexes (G4s) are DNA or RNA structures formed from stacked guanine tetrads. Computational studies have revealed the prevalence of more than 370,000 candidate putative. G-quadruplex DNA sequences (PQS) in various regions of the human genome [1]. The initial algorithm was designed to search for all stable G-quadruplexes with loop lengths up to 7 nt. The possibility of the formation of G4 structures possessing longer loops (L > 7–20) has been recently demonstrated [2,3,4,5]. The intramolecular base pairing in a long loop within the PQS was shown to be a driving force for the formation of stable quadruplex-duplex structures. The identification of over 80,000 stem loop-containing G-quadruplex sequences (SLQS) in the human genome suggests
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