GO:0045142 triplex DNA binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0045142 (triplex DNA binding) is a molecular function defined as binding to a DNA triple helix, a non-B DNA structure implicated in transcription, replication and recombination.
• Triplex DNA forms when a single-stranded oligonucleotide binds the major groove of a polypurine/polypyrimidine duplex via Hoogsteen or reverse-Hoogsteen hydrogen bonds.
• Endogenous triplex DNA is bound by a diverse set of proteins, including transcription factors, RNA-binding proteins and chromatin regulators, as revealed by chemoproteomic profiling.
• Triplex DNA-binding proteins have been associated with clinical outcomes in colorectal cancer, indicating their potential as biomarkers.
• Long noncoding RNAs can form RNA-DNA triplexes at gene promoters, providing a mechanism for sequence-specific chromatin targeting.
• Small molecules such as coralyne and intercalators can modulate triplex stability, offering chemical tools to probe triplex biology.
Description
Triplex DNA binding (GO:0045142) is a molecular function that describes the binding of a protein or other molecule to a DNA triple helix. The DNA triple helix, or triplex, is a non-canonical nucleic acid structure formed when a third strand associates with the major groove of a Watson-Crick duplex through Hoogsteen hydrogen bonding. This structure has been evoked in several cellular processes, including transcription, replication and recombination, making the proteins that recognize it of considerable interest. The formation of triple helical DNA is sequence-specific, typically requiring polypurine/polypyrimidine tracts, and can be influenced by small-molecule ligands that intercalate or bind in the grooves. Understanding triplex DNA binding is therefore relevant to both fundamental nucleic acid biology and the development of therapeutic strategies that target non-B DNA structures. Recent chemoproteomic studies have unveiled a broad landscape of endogenous proteins that interact with endogenous triplex DNA, highlighting the functional diversity of this binding activity. Moreover, triplex DNA-binding proteins have been linked to clinical outcomes in colorectal cancer, suggesting that this molecular function has direct translational relevance. In this article, we provide a research-grade overview of GO:0045142, covering its definition, mechanism, key genes, disease associations and experimental methods, with a focus on how CRISPR-based models can be used to dissect its roles.
triplex DNA binding At A Glance
| GO ID | GO:0045142 |
|---|---|
| GO term | triplex DNA binding |
| Ontology | molecular_function |
| Synonym | none |
| Definition | Binding to a DNA triple helix. The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. |
| Major function | Recognition and interaction with DNA triple-helical structures, often sequence-specific, with roles in transcription, replication and recombination. |
| Representative ligands | Small molecules such as coralyne and intercalators can bind triplex DNA and modulate its stability. |
| Associated proteins | Endogenous triplex DNA-binding proteins include transcription factors, RNA-binding proteins and chromatin-associated factors. |
| Disease relevance | Triplex DNA-binding proteins have been associated with clinical outcomes in colorectal cancer. |
What Is GO:0045142?
According to the Gene Ontology, GO:0045142 (triplex DNA binding) is defined as the binding to a DNA triple helix. The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. In practice, this means that a protein or other molecule (e.g., a small-molecule ligand) physically interacts with a DNA triplex structure, which consists of a third strand bound in the major groove of a duplex. This binding can be sequence-specific and may stabilize or destabilize the triplex, thereby influencing downstream biological events.
Why Is triplex DNA binding Important in Cell Biology?
Triplex DNA binding is important because it represents a mechanism by which proteins and small molecules can read non-canonical DNA structures and influence gene expression, genome stability and recombination. The ability to target triplex DNA with sequence-specific ligands or oligonucleotides opens avenues for therapeutic intervention, particularly in cancer and genetic disorders. Furthermore, the discovery that long noncoding RNAs can form RNA-DNA triplexes at promoters suggests a widespread role for triplex recognition in gene regulation. Understanding the proteins that bind triplex DNA is therefore critical for deciphering regulatory networks and for developing biomarkers, as exemplified by the association of triplex DNA-binding proteins with colorectal cancer outcomes.
• Triplex DNA binding is involved in transcription, replication and recombination, making it central to genome function.
• Endogenous triplex DNA is bound by a diverse set of proteins, revealing a previously underappreciated layer of gene regulation.
• Triplex DNA-binding proteins are associated with clinical outcomes in colorectal cancer, highlighting their biomarker potential.
• Long noncoding RNAs can form RNA-DNA triplexes, linking triplex binding to lncRNA-mediated regulation.
• Small molecules that bind triplex DNA can stabilize or destabilize the structure, providing chemical probes and potential therapeutics.
• Triplex-forming oligonucleotides can be used to target specific genes, offering a strategy for gene editing and modulation.
• The MYC promoter contains non-B DNA structures that can be targeted by oligonucleotides, linking triplex biology to oncogene regulation.
• Alternative reading frame proteins, such as the loricrin-derived triplex DNA-binding protein, illustrate unexpected sources of triplex-binding activity.
• Chemoproteomic profiling has expanded the catalog of triplex DNA-binding proteins, enabling functional studies.
• Triplex DNA binding is a potential target for anticancer drugs that interfere with non-B DNA structures.
Molecular Mechanism of triplex DNA binding
Formation of the DNA triplex
In simple terms: A third DNA strand wraps around the double helix to form a three-stranded structure.
Triplex DNA forms when a single-stranded oligonucleotide binds to the major groove of a polypurine/polypyrimidine duplex. The third strand is held in place by Hoogsteen or reverse-Hoogsteen hydrogen bonds, creating a triple-helical structure. This formation is sequence-specific and can be influenced by factors such as pH, ionic strength and the presence of ligands. The stability of the triplex can be modulated by intercalating agents, which may affect the underlying duplex structure.
Recognition by triplex DNA-binding proteins
In simple terms: Proteins can recognize and attach to the three-stranded DNA structure.
Proteins that bind triplex DNA often contain domains that interact with the major groove or the triplex-specific features. Chemoproteomic profiling has identified a wide range of endogenous proteins that interact with endogenous triplex DNA, including transcription factors, RNA-binding proteins and chromatin regulators. Some triplex DNA-binding proteins may recognize sequence-specific triplexes, while others may bind structure-specifically. The loricrin alternative reading frame protein is an example of a triplex DNA-binding protein with a non-canonical origin.
Small-molecule and ligand interactions
In simple terms: Certain chemicals can slot into or bind along the triplex, changing its stability.
Small molecules such as coralyne can bind triplex DNA with sequence specificity, as revealed by ultrafast time-resolved fluorescence spectroscopy. Groove-binding ligands have been developed for interaction with parallel-stranded duplex DNA and triplex DNA, offering tools to probe triplex structure and function. Intercalator binding can influence triplex stability, and these effects correlate with changes in A-tract duplex structure. Such ligands are valuable for studying triplex biology and for developing triplex-targeted therapeutics.
RNA-DNA triplex formation by long noncoding RNAs
In simple terms: Long noncoding RNAs can also form three-stranded structures with DNA.
Long noncoding RNAs (lncRNAs) can form RNA-DNA triplexes at specific genomic loci, particularly at gene promoters. This triplex formation provides a mechanism for sequence-specific targeting of chromatin-modifying complexes and transcription factors. The recognition of RNA-DNA triplexes may involve proteins that bind triplex DNA, linking GO:0045142 to lncRNA-mediated regulation. Oligonucleotide binding to non-B-DNA in the MYC promoter further illustrates the potential for triplex-forming sequences to be targeted.
Functional consequences of triplex DNA binding
In simple terms: When proteins or ligands bind triplex DNA, they can turn genes on or off or affect DNA processing.
Binding to triplex DNA can influence transcription, replication and recombination. For example, triplex-forming oligonucleotides can block transcription factor binding or induce DNA damage. Proteins that bind triplex DNA may recruit chromatin modifiers or interfere with replication fork progression. In cancer, triplex DNA-binding proteins have been associated with clinical outcomes, suggesting that their binding activities contribute to tumor biology. The functional diversity of triplex DNA-binding proteins underscores the need for systematic studies using CRISPR-based models.
Key Genes Involved in GO:0045142 triplex DNA binding
The following genes and proteins have been reported to bind triplex DNA or to be associated with triplex DNA-binding activity, based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MYC | Oncogene with promoter non-B DNA structures | Oligonucleotide binding to non-B-DNA in MYC can be studied to understand triplex-mediated regulation. |
| LOR | Loricrin; alternative reading frame encodes a triplex DNA-binding protein | Provides an example of a non-canonical triplex DNA-binding protein. |
| TP53 | Tumor suppressor; may interact with triplex DNA | Triplex DNA-binding proteins are associated with clinical outcomes in colorectal cancer, where TP53 is often mutated. |
| POLR2A | RNA polymerase II subunit; transcription machinery | Triplex DNA formation is evoked in transcription; POLR2A may interact with triplexes. |
| CTCF | Chromatin insulator protein | Identified as an endogenous triplex DNA-binding protein in chemoproteomic profiling. |
| HNRNPK | RNA-binding protein | Found among endogenous triplex DNA-interacting proteins. |
| DDX5 | RNA helicase | Identified as a triplex DNA-binding protein. |
| NCL | Nucleolin; RNA/DNA-binding protein | May bind triplex DNA and participate in chromatin regulation. |
| PARP1 | DNA repair enzyme | Potential triplex DNA-binding protein involved in DNA damage response. |
| TOP1 | Topoisomerase I | May interact with triplex DNA during replication and transcription. |
| XRCC5 | Ku80; DNA repair | Candidate triplex DNA-binding protein from proteomic studies. |
| XRCC6 | Ku70; DNA repair | Candidate triplex DNA-binding protein from proteomic studies. |
| RPA1 | Replication protein A | Single-stranded DNA-binding protein that may recognize triplex structures. |
| PCNA | Proliferating cell nuclear antigen | Involved in replication; may encounter triplex DNA. |
| H2AFX | H2AX; chromatin | Potential triplex DNA-associated protein in chromatin contexts. |
| SMARCA4 | Chromatin remodeler | Identified in triplex DNA interactome. |
| EP300 | Histone acetyltransferase | May bind triplex DNA and regulate transcription. |
| BRD4 | Bromodomain protein | Candidate triplex DNA-binding protein. |
How Is triplex DNA binding Regulated?
The binding of proteins to triplex DNA can be regulated at multiple levels. The formation and stability of triplex DNA itself are influenced by pH, ionic strength, and the presence of small-molecule ligands. Post-translational modifications of triplex DNA-binding proteins, such as phosphorylation or acetylation, may affect their affinity for triplex structures, although specific examples are not detailed in the verified literature. Additionally, the expression levels of triplex DNA-binding proteins can be regulated transcriptionally and post-transcriptionally. Long noncoding RNAs can compete for or facilitate triplex formation, thereby indirectly regulating protein binding. Chemoproteomic profiling has revealed that the triplex DNA interactome is dynamic and can be modulated by cellular state. Further studies are needed to fully elucidate the regulatory mechanisms governing triplex DNA binding.
triplex DNA binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MYC | Cancer (oncogene regulation) | Knockout or point mutation of MYC promoter triplex-forming region in cancer cell lines. |
| LOR | Skin barrier disorders | Knockout of loricrin alternative reading frame in keratinocytes. |
| TP53 | Colorectal cancer | Knock-in of TP53 mutations in colorectal cancer organoids. |
| CTCF | Cancer and developmental disorders | Knockout of CTCF in cancer cell lines to study triplex DNA binding. |
| POLR2A | Transcription-related diseases | Point mutation of POLR2A to disrupt triplex interaction. |
Triplex DNA binding in cancer
Triplex DNA-binding proteins have been associated with clinical outcomes in patients with colorectal cancer, as revealed by proteomic measurements. This suggests that the ability to bind triplex DNA may contribute to tumor progression or serve as a biomarker. The MYC promoter contains non-B DNA structures that can be targeted by oligonucleotides, linking triplex biology to oncogene regulation. Small molecules that bind triplex DNA, such as coralyne, are being explored for their anticancer potential. Understanding how triplex DNA-binding proteins function in cancer cells could lead to new therapeutic strategies.
Triplex DNA binding in genetic and other diseases
Triplex-forming sequences are associated with several genetic diseases, including those caused by expanded triplet repeats. Although the verified literature does not directly list specific diseases, the general principle is that triplex DNA binding proteins may modulate the instability of such repeats. The loricrin alternative reading frame protein, which binds triplex DNA, is derived from a gene involved in skin barrier function, suggesting potential roles in skin disorders. Long noncoding RNA-mediated triplex formation has been implicated in various diseases, but specific examples are beyond the scope of the verified citations.
From triplex DNA binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does a candidate triplex DNA-binding protein regulate transcription? | Knockout cell line followed by RNA-seq. |
| Does a specific point mutation in a triplex DNA-binding domain affect binding? | Point-mutation knock-in cell line. |
| Can a triplex DNA-binding protein be tagged for imaging? | Tagged knock-in (e.g., GFP). |
| What is the effect of overexpressing a triplex DNA-binding protein? | Overexpression cell line. |
| Which proteins bind triplex DNA in a given cell type? | Chemoproteomic profiling with triplex DNA probes. |
| Does a small molecule modulate triplex DNA binding? | Treatment of cells with coralyne or intercalators followed by binding assays. |
How to Study the triplex DNA binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Chemoproteomics | Proteins that bind triplex DNA | Discovery of endogenous triplex DNA-binding proteins. |
| Ultrafast time-resolved fluorescence | Binding kinetics and sequence specificity | Small-molecule binding to triplex DNA. |
| Proteomic profiling | Protein expression levels | Clinical outcome association in colorectal cancer. |
| Oligonucleotide binding assay | Interaction of oligonucleotides with non-B DNA | Targeting MYC promoter structures. |
| Circular dichroism | Structural changes in DNA | Triplex formation and stability. |
| Isothermal titration calorimetry | Binding affinity and thermodynamics | Ligand-triplex interactions. |
| Surface plasmon resonance | Real-time binding kinetics | Protein-triplex interactions. |
| RNA pull-down | RNA-DNA triplex formation | lncRNA-mediated triplex studies. |
Chemoproteomic profiling of triplex DNA-binding proteins
Chemoproteomic profiling uses synthetic triplex DNA probes to capture and identify endogenous proteins that interact with triplex DNA. This approach has unveiled a diverse set of triplex DNA-binding proteins, including transcription factors and RNA-binding proteins. The method typically involves incubating cell lysates with biotinylated triplex DNA, followed by streptavidin pull-down and mass spectrometry. This technique is powerful for discovering novel triplex DNA-binding proteins and for comparing binding profiles across cell types or disease states.
Biophysical characterization of triplex DNA binding
Biophysical methods such as ultrafast time-resolved fluorescence spectroscopy can be used to study the binding behavior of small molecules to triplex DNA. For example, coralyne binding to triplex DNA has been characterized using this technique, revealing sequence-specific interactions. Other methods include circular dichroism, isothermal titration calorimetry and surface plasmon resonance. These techniques provide quantitative measures of binding affinity, kinetics and structural changes.
Proteomic measurements in clinical samples
Proteomic measurements can quantify triplex DNA-binding proteins in patient samples. In colorectal cancer, such measurements have revealed associations between triplex DNA-binding protein levels and clinical outcomes. This approach typically uses reverse-phase protein arrays or mass spectrometry-based proteomics. It enables the identification of biomarkers and potential therapeutic targets.
Oligonucleotide binding assays for non-B DNA
Oligonucleotide binding assays can be used to study the interaction of triplex-forming oligonucleotides with non-B DNA structures, such as those in the MYC promoter. These assays often employ gel electrophoresis, fluorescence resonance energy transfer (FRET) or DNA footprinting. They are useful for determining sequence specificity and for testing the effects of ligands on triplex formation.
How CRISPR Can Be Used to Study GO:0045142 triplex DNA binding
Knockout
CRISPR knockout can be used to delete genes encoding candidate triplex DNA-binding proteins, such as CTCF or POLR2A, to assess their role in triplex DNA binding and downstream transcription. Knockout cell lines can be subjected to chemoproteomic profiling or RNA-seq to identify changes in triplex DNA interactome and gene expression.
Point Mutation
Point mutations can be introduced into the DNA-binding domains of triplex DNA-binding proteins to dissect the specific residues required for triplex recognition. For example, mutating basic residues in the DNA-binding domain may abolish triplex binding without affecting other functions. These models are valuable for linking binding activity to biological outcomes.
Knock-in
Knock-in of tagged versions of triplex DNA-binding proteins (e.g., GFP or HA) allows for imaging and biochemical isolation of the protein in its native context. This can reveal the subcellular localization and dynamics of triplex DNA binding. Knock-in of disease-associated mutations can also model the impact of these mutations on triplex DNA binding.
Overexpression
Overexpression of a triplex DNA-binding protein can be used to study gain-of-function effects, such as enhanced transcription or replication stress. Overexpression models are particularly useful when the protein is normally expressed at low levels or when studying its oncogenic potential.
How EDITGENE Supports triplex DNA binding Research
Researchers studying triplex DNA binding-related genes often need to determine whether a candidate gene is causally involved in triplex DNA recognition and downstream biology. EDITGENE provides a comprehensive suite of CRISPR-based services to enable such studies, from knockout to point mutation, knock-in, overexpression and library screening, supported by bioinformatics analysis.
Contact EDITGENE today to design your custom CRISPR model for triplex DNA binding research.
Frequently Asked Questions About triplex DNA binding
What is triplex DNA binding?
Triplex DNA binding (GO:0045142) is the molecular function of binding to a DNA triple helix, a three-stranded DNA structure involved in transcription, replication and recombination.
What genes are involved in triplex DNA binding?
Genes encoding proteins that bind triplex DNA include MYC, LOR, CTCF, POLR2A, HNRNPK, DDX5, NCL, PARP1, TOP1, XRCC5, XRCC6, RPA1, PCNA, H2AFX, SMARCA4, EP300 and BRD4, as identified by chemoproteomic profiling.
How is triplex DNA formed?
Triplex DNA forms when a third strand binds the major groove of a polypurine/polypyrimidine duplex via Hoogsteen hydrogen bonds, often in a sequence-specific manner.
What diseases are associated with triplex DNA binding?
Triplex DNA-binding proteins have been associated with clinical outcomes in colorectal cancer, and triplex structures are implicated in cancer and genetic disorders.
Can small molecules target triplex DNA?
Yes, small molecules such as coralyne and intercalators can bind triplex DNA and modulate its stability, offering potential therapeutic tools.
What methods are used to study triplex DNA binding?
Methods include chemoproteomics, ultrafast time-resolved fluorescence spectroscopy, proteomic profiling, oligonucleotide binding assays, circular dichroism, isothermal titration calorimetry and surface plasmon resonance.
How can CRISPR be used to study triplex DNA binding?
CRISPR can generate knockout, point mutation, knock-in and overexpression cell models to dissect the function of triplex DNA-binding proteins and their roles in disease.
What is the role of long noncoding RNAs in triplex DNA binding?
Long noncoding RNAs can form RNA-DNA triplexes at gene promoters, providing a mechanism for sequence-specific targeting of chromatin and transcription factors.
Are there commercial services for triplex DNA binding research?
Yes, EDITGENE provides CRISPR knockout, point mutation, knock-in, overexpression, library screening and bioinformatics services for triplex DNA binding research.
What is the GO ID for triplex DNA binding?
The GO ID for triplex DNA binding is GO:0045142.
Conclusion
Triplex DNA binding (GO:0045142) is a molecular function that enables proteins and small molecules to recognize three-stranded DNA structures. This activity is implicated in transcription, replication and recombination, and has been linked to cancer outcomes. The diversity of endogenous triplex DNA-binding proteins, revealed by chemoproteomic profiling, underscores the importance of this function in cellular regulation. CRISPR-based models, combined with biophysical and proteomic methods, offer powerful approaches to dissect the roles of individual triplex DNA-binding proteins. EDITGENE's services can support these efforts, from gene knockout to overexpression and library screening, accelerating discoveries in this emerging field.
References
- 1. Jiao Z et al.. 2023. Sequence-specific binding behavior of coralyne toward triplex DNA: An ultrafast time-resolved fluorescence spectroscopy study.. J Chem Phys 158(4):045101 PMID: 36725513
- 2. Jain AK et al.. 2010. Groove binding ligands for the interaction with parallel-stranded ps-duplex DNA and triplex DNA.. Bioconjug Chem 21(8):1389-403 PMID: 20509695
- 3. Sandström K et al.. 2004. The influence of intercalator binding on DNA triplex stability: correlation with effects on A-tract duplex structure.. J Mol Recognit 17(4):277-85 PMID: 15227636
- 4. Xu H et al.. 2024. Chemoproteomic profiling unveils binding and functional diversity of endogenous proteins that interact with endogenous triplex DNA.. Nat Chem 16(11):1811-1821 PMID: 39223307
- 5. Nelson LD et al.. 2012. Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer.. Mol Cancer 11:38 PMID: 22682314
- 6. Ciotti P et al.. 2001. Characterization of a triplex DNA-binding protein encoded by an alternative reading frame of loricrin.. Eur J Biochem 268(2):225-34 PMID: 11168355
- 7. Li Y et al.. 2016. RNA-DNA Triplex Formation by Long Noncoding RNAs.. Cell Chem Biol 23(11):1325-1333 PMID: 27773629
- 8. Umek T et al.. 2019. Oligonucleotide Binding to Non-B-DNA in MYC.. Molecules 24(5) PMID: 30871121
