Figure 3.

Electrophoretic mobility shift assays support triple helix formation. Triple helix formation between (A) HOTAIR novel TFD and HOXD3 and (B) TUG1 novel TFD and PPARGC1A, which were considered for in vitro validation. (C and D) Electrophoretic mobility shift assay of predicted binding domains (HOTAIR and TUG1). Complementary oligodeoxynucleotides were preincubated to form double stranded DNA and then incubated with either specific RNA of predicted triplex binding in HOTAIR (C) and TUG1 (D), or non-specific control RNA. RNA was applied in 25-fold or 50-fold excess; 1.1 equivalents were used of the pyrimidine-rich DNA strand to reduce the possibility of DNA:DNA-DNA triplex formation. A mobility shift that indicates triplex formation was only observed with the specific sequences of HOTAIR and TUG1 TFDs. Triplex formation increased wth the increased in concentrations of RNAs: HOTAIR (E) and TUG1 (F).