Modified RNA triplexes: Thermodynamics, structure and biological potential

Scientific Reports
Marta SzabatRyszard Kierzek

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 w...Continue Reading

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Methods Mentioned

BETA
circular dichroism
interaction studies
electrophoretic mobility shift assay
gel mobility shift
electrophoresis
transfection
PCR

Software Mentioned

Rad CFX Manager
MeltWin
Bio
Origin
OriginLab

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