Abstract
The occurrence of triplexes in vivo has been well documented and is determined by the presence of long homopurine-homopyrimidine tracts. The formation of these structures is the result of conformational changes that occur in the duplex, which allow the binding of a third strand within the major groove of the helix. Formation of these noncanonical forms by introducing synthetic triplex-forming oligonucleotides (TFOs) into the cell may have applications in molecular biology, diagnostics and therapy. This study focused on the formation of RNA triplexes as well as their thermal stability and biological potential in the HeLa cell line. Thermodynamics studies revealed that the incorporation of multiple locked nucleic acid (LNA) and 2-thiouridine (2-thioU) residues increased the stability of RNA triplexes. These data suggest that the number and position of the modified nucleotides within TFOs significantly stabilize the formed structures. Moreover, specificity of the interactions between the modified TFOs and the RNA hairpin was characterized using electrophoretic mobility-shift assay (EMSA), and triplex dissociation constants have been also determined. Finally, through quantitative analysis of GFP expression, the triplex structures were shown to regulate GFP gene silencing. Together, our data provide a first glimpse into the thermodynamic, structural and biological properties of LNA- and 2-thioU modified RNA triplexes.
Highlights
The occurrence of triplexes in vivo has been well documented and is determined by the presence of long homopurine-homopyrimidine tracts
Triplexes were first observed by Felsenfeld et al and occur when a triplex-forming oligonucleotide (TFO) binds in the major groove of DNA or RNA duplexes[1]
locked nucleic acid (LNA) appeared to be useful chemical tools to modulate the thermal stability of triple-helical structures[16,33]
Summary
Some papers concerning triplex-based strategies revealed interesting and exceptional properties of these structures in therapeutic contexts[30,31,32]. The incorporation of 2-thiouridine into the TFO strand has been shown to stabilize triplex formation with RNA and DNA duplex regions[3]. The influence of simultaneous LNA and 2-thioU substitutions within the TFO strand on triplex thermal stability. The thermodynamic data demonstrate that the simultaneous presence of LNA and 2-thioU modifications within the TFO strand has a significant influence on the thermal stabilities of T1–T8 model triplexes. The presence of LNA-cytidine and 2-thioU residues increased the melting temperatures of the RNA triplexes by 15.1 °C on average, in comparison to an unmodified counterpart (Table 1). The changes in melting temperatures correspond to the numbers of LNA and 2-thioU residues within the TFO strand This observation agrees with previous findings that showed the stabilization effect of LNA on triplex formation[16,34]. Previous papers revealed that the CD spectra of DNA triple-helical
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