Modified RNA triplexes: Thermodynamics, structure and biological potential

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.

Incorporating 2-Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A-U Pairs and for Targeting a MicroRNA Hairpin Precursor

“…Compared to T-to-U substitutions, T-to-s 2 U substitutions in PNAs may result in more significant strengthening of triplex formation, due to a reduced energy required for the dehydration, enhanced base stacking, in addition to a strengthened hydrogen bonding interaction. ,− Stacking interactions are often dependent on sequence context, which may result in sequence-dependent stabilization of T-to-s 2 U substitutions. We tested the effects of a single T-to-s 2 U substitution at varied positions [positions 1, 3, and 5–7 (Figure f and Table )] of PNA P1 on PNA·RNA 2 triplex formation.…”

“…Incorporation of s 2 U residues into a PNA is expected to stabilize a PNA–RNA duplex and a PNA·RNA–RNA triplex through the formation of stabilized Watson–Crick s 2 U-A pairs and s 2 U·A-U triples, respectively (Figure e,f). − However, incorporation of L residues into PNAs stabilizes the PNA·RNA–RNA triplex and destabilizes the PNA–RNA duplex, with the formation of stable L·G-C triples and unstable L-G Watson–Crick pairs (Figure c,d) . Thus, it is important to test if PNAs containing s 2 U and L residues (Figure c,e) still show the selective recognition of dsRNAs over ssRNAs.…”

“…Incorporation of 2-thiouracil (s 2 U) into oligonucleotides stabilizes the formation of duplexes and triplexes through the formation of the Watson–Crick s 2 U-A base pair and s 2 U·A-U base triple, respectively (Figure e,f). ,− An s 2 U modification in oligonucleotides favors C3′-endo sugar pucker induced by a steric effect between the 2-thiocarbonyl group and the 2′-hydroxyl group . In addition, thiolation of uracil causes a decrease in the dehydration energy penalty as well as a decrease in the p K a of the N3 atom, which may result in an improved hydrogen bonding involving imino proton H3 in s 2 U. ,,, The enhanced stacking interaction of the 2-thiocarbonyl group with adjacent bases may also contribute to the formation of stable TFO·RNA 2 triplexes. , …”

Synthesis and surface characterization of new triplex polymer of Ag(I) and mixture nucleosides: cytidine and 8-bromoguanosine

“…Substitution C8 in guanosine [13, 15, 29, 30] molecular in Y motif can increase the stability of the triplex structure in addition to the stability that produces from the presence of Ag(I) ions and the influence of Hoogsteen hydrogen bonding in this structure. Most triplexes structures that concern RNA are synthesised regarding functionally RNAs such as ribosomal RNAs [31], telomerase RNAs [32], and long noncoding RNAs [33, 34]. In addition, these materials played a great role in molecular biology [35] and nanotechnology applications [36, 37].…”

RNA triplex structures revealed by WAXS-driven MD simulations

“…We designed an RNA hairpin construct called ”Short-Tetraloop Duplex” (ST-DPX), consisting of a UUCG tetraloop and a duplex stem with 17 base pairs: 23, 68, 69 5’- GGG CUA GAG AGA GAA AGU UUC GAC UUU CUC UCU CUA GCC C. The duplex stem further contains a 12-base-paired TFO12 binding domain, designed to form 12 consecutively stacked base triples, followed by 4 GC base pairs at the closing (blunt) end. The 12-base-paired TFO binding domain is an asymmetric sequence to ensure a specific binding direction of the partner TFO12; reverse binding of TFO12 would disrupt the consecutive formation of base triples and therefore reduce the stability of the RNA triplex construct.…”