Stability of an RNA•DNA-DNA triple helix depends on base triplet composition and length of the RNA third strand.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA-DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA-DNA triple helices is not well understood beyond the canonical U•A-T and C•G-C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), in an RNA•DNA-DNA triple helix was determined. The canonical U•A-T and C•G-C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA-DNA triple helix can tolerate up to two consecutive non-canonical A•G-C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA-DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA-DNA and RNA•RNA-RNA triple helices was distinctly different from those in RNA•DNA-DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.