“…They are nontoxic, penetrate into living cells and reach their targets in nuclei. [6][7][8][9] In this sense, they have many advantages in comparison to triplexforming oligonucleotides, 10 another class of relatively small dsDNA-recognizing molecules that were used in TISH 11 and COMBO-FISH 12,13 approaches. Coupling of several polyamides into tandem structures increases the length of recognition sequence, thus improving their specificity.…”
“…They are nontoxic, penetrate into living cells and reach their targets in nuclei. [6][7][8][9] In this sense, they have many advantages in comparison to triplexforming oligonucleotides, 10 another class of relatively small dsDNA-recognizing molecules that were used in TISH 11 and COMBO-FISH 12,13 approaches. Coupling of several polyamides into tandem structures increases the length of recognition sequence, thus improving their specificity.…”
“…This mechanism could be conserved across mammals by conserving r-AG motifs in L1s by convergence in nucleotide variations and by maintaining particular r-UC/r-AG motif ratios in the involved lncRNAs, despite their poorly conserved sequences. Future efforts will focus on characterizing the functions of short UC (TC)/AG-motifs as well as other triple-helix motifs such as (G, T)-motifs [37, 74] to further aid our understanding of the redundant mechanisms of lncRNA–chromatin associations in the XCI process.…”
Section: Discussionmentioning
confidence: 99%
“…We also showed convergent patterns of variation in L1 base composition, which results in the conservation of the short motifs in L1s, thereby contributing to the retention of their ability across mammalian species to redundantly form triplexes with the lncRNAs involved in XCI, these occurring through Hoogsteen (the third strand pyrimidine in parallel orientation) or reverse Hoogsteen (the third strand purine in antiparallel orientation) base-pairing (Fig. 1; [37]).Fig. 1Schematic representation of triple helices.…”
Section: Introductionmentioning
confidence: 89%
“…To investigate the features of short motifs, we used the reference sequences of XIST/Xist/Rsx (NR_001564/NR_001463/JQ937282), human lncRNAs (RNAcentral), and LINEs on the X and other chromosomes (NCBI hg38 for human/mm10 for mouse/monDom5 for opossum) according to RepeatMasker annotations. For performing the searches of the number, location, length-distribution, and ratio of r-UC (TC)/r-AG motifs, we used tailor-made software (DNAMotifFinder) that can set the search parameters (minimum length and number of consecutive nucleotides in indicated situations) according to the Hoogsteen or reverse Hoogsteen base-pairing rules [37, 38]. For mouse and human LINEs, the parameters were subject to the conditions suitable for Hoogsteen base-paring with XIST/Xist RNAs, which predominantly contain r-UC motifs, whereas, for opossum LINEs, the parameters were subject to the conditions suitable for reverse Hoogsteen base-paring with Rsx RNA, where r-AG motifs are predominant.…”
Background
Associations between X-inactive transcript (Xist)–long noncoding RNA (lncRNA) and chromatin are critical intermolecular interactions in the X-chromosome inactivation (XCI) process. Despite high-resolution analyses of the Xist RNA-binding sites, specific interaction sequences are yet to be identified. Based on elusive features of the association between Xist RNA and chromatin and the possible existence of multiple low-affinity binding sites in Xist RNA, we defined short motifs (≥5 nucleotides), termed as redundant UC/TC (r-UC/TC) or AG (r-AG) motifs, which may help in the mediation of triplex formation between the lncRNAs and duplex DNA.
Results
The study showed that r-UC motifs are densely dispersed throughout mouse and human Xist/XIST RNAs, whereas r-AG motifs are even more densely dispersed along opossum RNA-on-the-silent X (Rsx) RNA, and also along both full-length and truncated long interspersed nuclear elements (LINE-1s, L1s) of the three species. Predicted secondary structures of the lncRNAs showed that the length range of these sequence motifs available for forming triplexes was even shorter, mainly 5- to 9-nucleotides long. Quartz crystal microbalance (QCM) measurements and Monte Carlo (MC) simulations indicated that minimum-length motifs can reinforce the binding state by increasing the copy number of the motifs in the same RNA or DNA molecule. Further, r-AG motifs in L1s had a similar length-distribution pattern, regardless of the similarities in the length or sequence of L1s across the three species; this also applies to high-frequency mutations in r-AG motifs, which suggests convergence in L1 sequence variations.
Conclusions
Multiple short motifs in both RNA and duplex DNA molecules could be brought together to form triplexes with either Hoogsteen or reverse Hoogsteen hydrogen bonding, by which their associations are cooperatively enhanced. This novel triplex interaction could be involved in associations between lncRNA and chromatin in XCI, particularly at the sites of L1s. Potential binding of Xist/XIST/Rsx RNAs specifically at L1s is most likely preserved through the r-AG motifs conserved in mammalian L1s through convergence in L1 nucleotide variations and by maintaining a particular r-UC/r-AG motif ratio in each of these lncRNAs, irrespective of their poorly conserved sequences.
Electronic supplementary material
The online version of this article (10.1186/s13100-019-0173-4) contains supplementary material, which is available to authorized users.
“…The recognition and detection of specific sequences in native genomic double-stranded DNA (dsDNA) is of significant importance for the development of efficient gene therapies and in vivo gene labeling [ 1 – 3 ]. Besides natural and engineered peptides or proteins, two synthetic substances are known to recognize and bind dsDNA in a sequence-specific manner: triplex-forming oligonucleotides (TFOs) [ 4 – 5 ] and pyrrole–imidazole polyamide minor groove binders (MGBs) [ 6 – 7 ]. TFOs recognize polypurine stretches in genomic DNA and bind in the major groove of dsDNA.…”
SummaryEfficient protocols based on Cu(I)-catalyzed azide–alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole–imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these conjugates did not affect the binding parameters (affinity and triplex stability) of the parent TFOs.
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