An Oligo-Library-Based Approach for Mapping DNA–DNA Triplex Interactions In Vitro

Abstract

We present Triplex-seq, a deep-sequencing method that systematically maps the interaction space between an oligo library of ssDNA triplex-forming oligos (TFOs) and a particular dsDNA triplex target site (TTS). We demonstrate the method using a randomized oligo library comprising 67 million variants, with five TTSs that differ in guanine (G) content, at two different buffer conditions, denoted pH 5 and pH 7. Our results show that G-rich triplexes form at both pH 5 and pH 7, with the pH 5 set being more stable, indicating that there is a subset of TFOs that form triplexes only at pH 5. In addition, using information analysis, we identify triplex-forming motifs (TFMs), which correspond to minimal functional TFO sequences. We demonstrate, in single-variant verification experiments, that TFOs with these TFMs indeed form a triplex with G-rich TTSs, and that a single mutation in the TFM motif can alleviate binding. Our results show that deepsequencing platforms can substantially expand our understanding of triplex binding rules and aid in refining the DNA triplex code.

TripDesign: A DNA Triplex Design Approach Based on Interaction Forces

“…= P Calculate(SubPop) fitness (10) Calculate (X) represents the stacking force of the sequence, and SubPop is a population with a size of 2 L , where L represents the length of the TFO.…”

“…In contrast, with other Web service tools, users must investigate potential off-target sites on a candidate-by-candidate basis. , In addition to this, some researchers have used sequencing technologies to provide triplex target sites (TTS) in specific genomes and systematically map the interaction space between TFOs and TTS. The formation of the DNA triplex provides a method for targeting gene sequences and holds great potential in the field of gene therapy. , At present, the following two strategies are usually adopted to improve the quality of the triplex. The first strategy is to add nucleic acid analogues such as bridged nucleic acids (BNA), fisetin (FIS), cationic silver nanoclusters (AgNCs), and other unnatural modifications to the triplex sequence.…”

“…The formation of the DNA triplex provides a method for targeting gene sequences and holds great potential in the field of gene therapy. 10,15 At present, the following two strategies are usually adopted to improve the quality of the triplex. The first strategy is to add nucleic acid analogues such as bridged nucleic acids (BNA), 16 fisetin (FIS), 17 cationic silver nanoclusters (AgNCs), 18 and other unnatural modifications to the triplex sequence.…”

“…As with Watson–Crick base pairing, Hoogsteen base pairing is guided by specific binding rules . To better understand the underlying binding rules, Buske et al developed the Triplexator platform, a computational toolkit for querying nucleic acid triplet formation .…”

“…As with Watson−Crick base pairing, Hoogsteen base pairing is guided by specific binding rules. 10 To better understand the underlying binding rules, Buske et al developed the Triplexator platform, a computational toolkit for querying nucleic acid triplet formation. 11 Subsequently, the team developed the Triplex-Inspector, a computational tool for evaluating the accuracy of triplex-based genomic manipulation, which can be used for sequence-specific ligand design and appropriate target selection.…”