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

Abstract

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.

Implication of repeat insertion domains in the trans-activity of the long non-coding RNA ANRIL

“…This supports the emergent idea that G/A-rich sequences might serve as anchoring motifs to direct lncRNAs toward specific genomic loci ( 85 , 86 ). We can speculate that such composition may favor triplex formation since G/A residues promote the most stable Hoogsteen base-pairing formation ( 87 ). Note that 13% of the ChIRP-peaks have a TTS for which no significant enrichment was found neither for the up- and -down regulated primary trans- targets nor for any DNA classes ( Supplementary Figure S7C ).…”

Unraveling the structure and biological functions of RNA triple helices

“…For intermolecular triple helices like lncRNA•gDNA, computational programs rely on Hoogsteen base‐pairing rules or the so‐called triple‐strand code. The relative stability of all 16 base triples has been quantitatively determined for D•D‐D, R•R‐R, and R•D‐D triple helices, although no universal rules emerge, at least for the 14 noncanonical base triples (Best & Dervan, 1995; Brown et al, 2016a; Kunkler et al, 2019; Mergny et al, 1991). Thus, a new experimental strategy to map R•D‐D triple helices inside cells has been developed, whereby protein‐free DNA‐associated RNA and RNA‐associated DNA is isolated and then subjected to a series of nuclease digests followed by high‐throughput sequencing (Senturk Cetin et al, 2019).…”

“…In the context of a U•A‐U‐rich major‐groove RNA triple helix, systematic studies are gradually being conducted to better understand the stability of noncanonical RNA base triples, which is any base triple other than U•A‐U or C + •G‐C. Here, the nucleotide composition of a single base triple is varied within a naturally occurring MALAT1 RNA triple helix (Brown et al, 2016a) or non‐naturally occurring RNA triple helix (Kunkler et al, 2019). These studies employed native gel‐shift assays and a cell‐based intronless β‐globin reporter assay to measure the relative stability of base triples.…”

Structural Recognition of Triple-Stranded DNA by Surface-Enhanced Raman Spectroscopy

“…As a model intermolecular triple-helix ( Figure 2 A), we selected one based on the association of a 31-base pair duplex (D 1 D 2 ), comprising a pyrimidine-rich strand (D 1 ) and a purine-rich strand (D 2 ), with a 22-nucleotide pyrimidine-rich strand (TFO). The third strand binds via formation of 19 T•AT and 2 C•GC base triplets adopting a parallel orientation along the major groove of the purine-rich D 2 strand in the Watson–Crick D 1 D 2 duplex [ 31 ]. The resulting D 1 D 2 •TFO construct (1:1:1 ratio, where “•” indicates the Hoogsteen interactions) has been previously characterized by conventional techniques, demonstrating to possess high stability at room temperature in a pseudo-physiological triplex buffer (pH 7, 120 mM NaCl, 8 mM Mg 2+ ) [ 31 ].…”

“…The third strand binds via formation of 19 T•AT and 2 C•GC base triplets adopting a parallel orientation along the major groove of the purine-rich D 2 strand in the Watson–Crick D 1 D 2 duplex [ 31 ]. The resulting D 1 D 2 •TFO construct (1:1:1 ratio, where “•” indicates the Hoogsteen interactions) has been previously characterized by conventional techniques, demonstrating to possess high stability at room temperature in a pseudo-physiological triplex buffer (pH 7, 120 mM NaCl, 8 mM Mg 2+ ) [ 31 ]. The triplex construct was obtained by combining an equimolar amount of TFO and D 1 D 2 duplex for 24 h at 4 °C.…”