The 5’UTR-dependent enhancement of protein translation efficiency triggered by self-transfecting 3’-aminoallyl-containing oligonucleotides (aa-dGoligos) targeting a pool of strongly folded transcript variants of the THRB suppressor gene

Background: The frequently reported lack of correlation between mRNA and protein levels of the human thyroid hormone receptor beta (THRB) suggests that translation of this suppressor is a mechanism emerging as an important target for control of the gene. In our previous studies, we have reported that microRNAs and siRNAs can inhibit the TRb1 expression. Recent studies have shown that interfering RNAs may also activate gene transcription. Thus far, the small activating RNAs (saRNAs) have only been shown as promoter-specific transcriptional activators. Purpose: The aim of this study was to develop a strategy to selectively enhance the expression of a target gene at the level of protein translation. Methods: We used oligonucleotides, designed to recognize sequence-specific targets within the weakly and strongly folded TRb1 5’UTRs variants A (vA) and F (vF), cloned into luciferase expressing plasmids. The binding sites were predicted using a calculator determining sequences, in which a single nucleotide substitution (in silico) was able to weaken the folding Gibb’s energy. Then, we synthesized a set of single-stranded DNA- and 2’-O-metyl-RNA-based oligonucleotides (termed dGoligos, dGs) as well as 3’-aminoallyl- derivatives (aa-dGs), prepared by incorporation of aminoallyl-dUTPs into 3’-end of the dGs followed by their coupling to positively charged fluorescent dyes (Cy3 or Cy5), facilitating the self-transfecting properties of the aa-dGs. Findings: Studies with an in vitro cell-free system revealed that the most weakly folded vA mediated 5.96-fold higher basal translation of luciferase when compared to the most strongly folded vF. The use of DNA-based antisense dG8 allowed for 6.0- (vA) and 8.3-fold (vF) protein overexpression, whereas a coupled action of dG8 and sense dG1 enhanced luciferase activity over 28.1- (vA) and 55.8-fold (vF). The in vivo PEI-based transfection of Caki-2 with vA-construct revealed that among cotransfected dGs the microRNA-like 2’-O-metyl-RNA based oligos were the most efficient enhancers. The improvement in translation was statistically significant for both micro-dG2 (1.7-fold) and -dG4 (and 2.6-fold). To obtain the same results of translation enhancement, 6.7-fold lower concentration of the self-transfecting aa-dG were needed. Significance: This is the first report showing a simple method for gene-specific translation enhancement.