Purine- and pyrimidine-triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus.

Rodrigo Maldonado,Michael Filarsky,Ingrid Grummt,Gernot Längst

doi:10.1261/rna.063800.117

Abstract

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA–DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G–C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.

Highlights

Triplexes are noncanonical DNA structures comprising an additional single-stranded RNA or DNA-binding sequence specific to the major groove of double-stranded DNA
We used Triplexator-predicted targeting sites (TTS) motifs stable, parallel Hoogsteen interactions requires the presence from human and mouse rDNA to study the stability of triplex of divalent cations, e.g., Mg2+, and/or slightly acidic pH for structures and their potential to form at physiological condiprotonation of cytosines in the triplex-forming oligonucleotides (TFOs) (Sugimoto et al 2001; tions (Fig. 1B)
We show that triplexes spect to the polypurine strand, potentially due to the right- containing an RNA TFO are more stable than DNA TFOs

Summary

Introduction

Single-stranded oligonucleotides with the potential to form triple helices, termed triplex-forming oligonucleotides (TFOs), bind to the major groove of DNA and interact with polypurine sequences through Hoogsteen base-pairing (Hoogsteen 1959; Rajagopal and Feignon 1989) (Fig. 1A). We used Triplexator-predicted TTS motifs stable, parallel Hoogsteen interactions requires the presence from human and mouse rDNA to study the stability of triplex of divalent cations, e.g., Mg2+, and/or slightly acidic pH for structures and their potential to form at physiological condiprotonation of cytosines in the TFO (Sugimoto et al 2001; tions (Fig. 1B).

Results

Conclusion