TL;DR:

Molecular interaction mapping reveals how proteins, DNA, RNA, and peptides interact across the genome. By combining technologies such as ChIP-seq, CUT&Tag, RIP-seq, eCLIP-seq, Hi-C, IP-MS, and RIME MS with tailored bioinformatics, epigenomic sequencing services can move from single-interaction validation to network-level insights that support mechanism-focused research.

Why Do Molecular Interactions Matter in Epigenomic Research

Molecular interactions are the physical contacts between biomolecules such as proteins, DNA, RNA, and peptides.

These contacts create layered regulatory networks that shape gene expression, chromatin structure, and cell identity.

Key interaction types include:

• Protein–DNA: transcription factors, histones, and DNA repair proteins binding genomic DNA
• Protein–RNA: RNA-binding proteins associating with mRNA and non-coding RNA
• DNA–DNA: distant genomic regions contacting each other in three-dimensional space
• Protein–protein: signaling complexes and chromatin-associated assemblies

Traditional co-immunoprecipitation (Co-IP) assays validate one interaction at a time.

High-throughput sequencing and mass spectrometry now enable "one-to-many" and even genome- or proteome-wide interaction maps in a single project.

For epigenomic research, this means:

• Systematic maps of regulatory factor binding
• 3D chromatin architecture profiling
• Network-level views of chromatin complexes and signaling pathways

How Do High-Throughput Interaction Technologies Work

High-throughput interaction technologies follow a shared logic: stabilize complexes, enrich targets, then read them out.

Most workflows include three core steps:

1. Capture interaction complexes
   • Crosslink or stabilize interactions in intact cells or tissue.
   • Use antibodies or affinity tags to pull down the complexes.
2. Isolate the relevant molecules
   • Purify bound DNA, RNA, or proteins.
   • Optionally fragment, ligate, or label the molecules.
3. Read and analyze
   • Use next-generation sequencing (NGS) or LC-MS/MS.
   • Map reads or peptides back to the genome or proteome.

The exact protocol depends on which interaction type you need to study.

What are Protein–DNA Interactions and How Do We Map Them

Protein–DNA interactions show where transcription factors, histone marks, and other chromatin proteins bind across the genome.

These binding patterns act as the "on/off switches" of gene regulation.

ChIP-seq: Classic Genome-Wide Binding Maps

ChIP-seq (chromatin immunoprecipitation sequencing) is a genome-wide method to map protein–DNA binding sites in vivo.

ChIP-seq is a sequencing-based assay that immunoprecipitates protein–DNA complexes and identifies their genomic binding sites.

Principle in brief

• Crosslink proteins to DNA in living cells, usually using formaldehyde.
• Fragment chromatin to 200–500 bp by sonication.
• Use a specific antibody to immunoprecipitate the protein–DNA complexes.
• Reverse crosslinks, purify DNA, and perform high-throughput sequencing.
• Align reads to the reference genome and call peaks to pinpoint binding sites.

Key characteristics

• Genome-wide and unbiased, no need to pre-define candidate regions.
• Typical resolution is around 50–100 bp, depending on fragment size and depth.
• Strongly depends on antibody specificity and crosslinking optimization.

Typical applications

• Identifying transcription factor binding sites under different conditions.
• Mapping histone modifications such as H3K4me3 or H3K27me3.
• Profiling chromatin-associated enzymes and repair factors.

CUT&Tag: Low-Input Alternative to ChIP-seq

CUT&Tag (cleavage under targets and tagmentation) is a tethered transposase method that maps protein–DNA binding with high efficiency and very low input.

CUT&Tag is an in situ assay in which an antibody-guided Tn5 transposase cuts and tags DNA only near the target protein, enabling direct sequencing of bound regions.

Principle in brief

• Bind a primary antibody to the target chromatin protein in intact cells or nuclei.
• Add a fusion of protein A/G and Tn5 transposase pre-loaded with sequencing adapters.
• Recruit Tn5 to antibody-bound sites, where it cuts and inserts adapters nearby.
• Purify tagged fragments and sequence them directly, without bulk sonication.

Key characteristics

• Extremely low input; often compatible with as few as 10²–10⁴ cells.
• Shorter workflow with fewer washes and lower background.
• Often somewhat less demanding on antibody performance than classic ChIP-seq.

Comparison of CUT&Tag, CUT&RUN and ChIP-seq showing high reproducibility and efficient peak detection across histone marks, even at lower read depths. (Kaya-Okur H.S. et al. (2019) Nature Communications)

Typical applications

• Protein–DNA mapping in rare or precious samples, including some clinical research biopsies.
• Multi-omics designs where chromatin binding is profiled alongside RNA-seq or ATAC-seq.

ChIP-seq vs CUT&Tag at a glance

How Do We Study Protein–RNA Interactions?

Protein–RNA interactions connect RNA-binding proteins (RBPs) with their target transcripts.

They control RNA splicing, localization, translation, and degradation.

Because RNA is fragile, most methods use UV crosslinking to stabilize RBP–RNA complexes inside cells before purification.

RIP-seq: Global Screening of RNA Targets

RIP-seq (RNA immunoprecipitation sequencing) is a transcriptome-wide method to identify RNAs associated with a given RBP.

RIP-seq is a sequencing assay that immunoprecipitates an RNA-binding protein and profiles the RNAs bound to it.

Principle in brief

• Use UV light to crosslink RBP–RNA complexes in cells.
• Lyse cells and perform immunoprecipitation with an antibody against the RBP.
• Reverse crosslinks, purify RNA, convert to cDNA, and sequence.
• Map reads to identify enriched transcripts associated with the protein.

Key characteristics

• Conceptually simple with fewer enzymatic steps.
• Modest positional resolution; it reveals target RNAs but not precise nucleotides.
• Non-specific RNA can co-purify, so strong controls are essential.
• Enables transcriptome-wide screening of candidate targets.

Typical applications

• Defining the RNA target set of an RBP in a given cell type.
• Comparing RBP-bound transcriptomes between healthy and disease models.

eCLIP-seq: High-Resolution RBP Binding Maps

eCLIP-seq (enhanced crosslinking and immunoprecipitation sequencing) is an optimized CLIP protocol that yields near nucleotide-level binding maps with lower input.

eCLIP-seq is a UV-crosslink-based method that enriches RBP–RNA complexes and uses characteristic mutation signatures to pinpoint binding sites at high resolution.

Principle in brief

• Irradiate cells at 254 nm to create covalent bonds at RBP–RNA contact points.
• Immunoprecipitate RBP–RNA complexes with a specific antibody.
• Use stringent washes to remove unbound RNA and proteins.
• Partially digest RNA into short fragments, typically 20–50 nucleotides.
• Ligate adapters in a controlled, stepwise fashion to reduce adapter contamination.
• Reverse transcribe; crosslink sites often introduce characteristic substitutions.
• Sequence and integrate mutation signatures with peak calling to map binding sites.

Key characteristics

• Low input requirement, often 5×10⁵–1×10⁶ cells per IP.
• Reduced adapter and background contamination compared with early CLIP protocols.
• High reproducibility and well suited to standardized pipelines.
• Combines global target discovery with near nucleotide-level positional information.

Typical applications

• Building high-resolution RBP interaction maps in disease models.
• Studying alternative splicing by linking RBP binding to exon usage.
• Exploring long non-coding RNA mechanisms through their protein partners.

RIP-seq vs eCLIP-seq at a glance

What Is Hi-C and How Does It Reveal 3D Genome Structure?

Hi-C is the foundational method for studying genome-wide DNA–DNA contacts and three-dimensional chromatin architecture.

Hi-C is a chromosome conformation capture sequencing method that detects physical proximity between genomic regions across the entire genome.

Principle in brief

• Crosslink chromatin in intact cells using formaldehyde to "freeze" 3D contacts.
• Digest DNA with a restriction enzyme to create sticky ends.
• Fill in ends and incorporate biotinylated nucleotides.
• Ligate DNA fragments so that spatially neighbouring pieces join into chimeric molecules.
• Shear DNA, enrich biotin-labeled ligation products, and perform paired-end sequencing.
• Map read pairs back to the genome and build a contact matrix.

Example Hi-C analysis workflow and contact map visualization showing genome-wide interaction matrices and statistically significant chromatin contacts. (Sikdar S. et al. (2020) BMC Genomics)

Key characteristics

• Genome-wide, unbiased view of chromatin contacts.
• Resolution depends strongly on sequencing depth; Mb-scale is common, kb-scale needs deep coverage.
• Captures interactions both within and between chromosomes.

Typical applications

• Tracking rewiring of chromatin domains during differentiation or reprogramming.
• Studying structural changes associated with cancer-related rearrangements.
• Defining topologically associating domains (TADs) and enhancer–promoter loops.

How Can We Map Protein–Protein Interaction Networks

Protein–protein interactions organize signaling cascades, chromatin complexes, and many epigenetic regulators.

Combining immunoprecipitation or affinity purification with mass spectrometry enables global screening of interaction partners.

IP-MS: Immunoprecipitation Coupled with Mass Spectrometry

IP-MS is a widely used workflow that combines antibody-based pull-down with LC-MS/MS readout.

IP-MS is a proteomics method that enriches a target protein and its partners using an antibody, then identifies all bound proteins by mass spectrometry.

Principle in brief

• Use a specific antibody to immunoprecipitate the protein of interest from lysates.
• Co-purify endogenous interaction partners as complexes.
• Digest proteins into peptides and analyze them by LC-MS/MS.
• Search spectra against protein databases to identify and quantify interactors.

Key characteristics

• Unbiased partner discovery without pre-defining candidates.
• Reflects in-cell interactions under near-physiological conditions.
• Strongly dependent on antibody specificity and mass spectrometer performance.

Typical applications

• Characterizing the interaction network of signaling or chromatin regulators.
• Comparing interaction landscapes between control and perturbed conditions.
• Defining the composition of multi-protein complexes.

RIME MS: Interaction Mapping for Nuclear and Chromatin Proteins

RIME MS (rapid immunoprecipitation mass spectrometry of endogenous proteins) is optimized for nuclear and chromatin-associated factors.

RIME MS is a fast IP-MS protocol that preserves transient and chromatin-confined interactions of nuclear proteins, including transcription factors.

Principle in brief

• Rapidly process fresh cells or tissues to minimize complex dissociation.
• Capture endogenous nuclear proteins and partners using a specific antibody.
• Enrich complexes with magnetic beads and perform stringent washes.
• Digest proteins on-bead and analyze by LC-MS/MS.
• Filter results using appropriate negative controls.

Key characteristics

• Preserves native interactions, including transient and low-abundance complexes.
• Often shows lower background than standard IP-MS for nuclear targets.
• Sensitive enough for fmol-level transcription factors.
• Compatible with cultured cells, fresh tissues, and some clinical research samples.

Typical applications

• Dissecting chromatin modifier complexes and associated cofactors.
• Capturing stimulus-dependent interaction changes in signaling pathways.
• Mapping oncogenic protein interaction networks and assessing drug effects on complexes.

TAP-MS: Tandem Affinity Purification with Mass Spectrometry

TAP-MS (tandem affinity purification mass spectrometry) uses engineered tags to achieve highly specific complex purification.

TAP-MS is a proteomics method where a bait protein is fused to two affinity tags and purified in two sequential steps before MS analysis.

Principle in brief

• Express a fusion of the protein of interest with two affinity tags.
• Perform the first affinity capture and elute the complexes gently.
• Apply a second affinity purification step to remove remaining contaminants.
• Digest the purified complexes and analyze by LC-MS/MS.

Key characteristics

• Very high specificity, yielding low background and high-confidence interactors.
• Detects weak and low-abundance partners effectively.
• Requires stable expression of tagged proteins, which may limit use in primary samples.

Typical applications

• Systematic mapping of core complex composition in model systems.
• Building reference interaction networks for specific pathways or organelles.

Y2H-seq: Yeast Two-Hybrid with Sequencing Readout

Y2H-seq extends traditional yeast two-hybrid screening with high-throughput sequencing.

Y2H-seq is a yeast-based protein–protein interaction screen where prey partners are identified by sequencing rather than colony picking.

Principle in brief

• Clone the protein of interest as a "bait" fusion in yeast.
• Express a cDNA library as "prey" fusions.
• Only yeast with interacting bait–prey pairs activate a reporter gene and survive selection.
• Sequence prey inserts from surviving colonies to identify interacting proteins.

Key characteristics

• High-throughput screen testing thousands of potential partners in parallel.
• Performed in yeast, which may differ from mammalian cellular context.
• Can generate false positives; biochemical validation is recommended.

Typical applications

• Initial discovery of potential interactors for novel proteins.
• Large-scale network mapping in basic research projects.

CLIP-MS: Crosslinking IP Combined with Mass Spectrometry

CLIP-MS uses chemical crosslinkers to stabilize transient complexes before IP-MS.

CLIP-MS is an IP-MS variant where crosslinking reagents capture weak or short-lived protein–protein interactions for proteomic identification.

Principle in brief

• Treat cells with a membrane-permeable crosslinker to lock interacting proteins together.
• Immunoprecipitate the protein of interest and its crosslinked partners.
• Digest complexes and analyze peptides by LC-MS/MS.

Key characteristics

• Enhances detection of transient and weak interactions.
• Crosslinking can introduce non-specific associations, so strict controls are essential.

Typical applications

• Mapping rapid signaling events downstream of stimuli.
• Studying cell cycle-dependent interaction networks.

Phage Display-seq: Peptide and Protein Binding Landscapes

Phage display-seq interrogates large peptide or protein fragment libraries for binding to a target.

Phage display-seq is a selection and sequencing method that identifies peptides or protein fragments binding a target protein using phage display and NGS.

Principle in brief

• Display diverse peptides or protein fragments on phage surfaces.
• Incubate the library with an immobilized target protein.
• Wash away non-binding phage; elute and amplify bound phage.
• Sequence displayed inserts to identify enriched binding motifs.

Key characteristics

• Extremely high diversity, often 10⁸–10¹⁰ unique sequences.
• Ideal for discovering binding motifs, epitopes or candidate inhibitors.
• Performed in vitro; physiological validation is required.

Typical applications

• Mapping binding motifs of receptors, enzymes or antibodies.
• Discovering candidate peptides for inhibitor or binder development.

How Do You Choose the Right Molecular Interaction Technology

No single assay answers every question about molecular interactions.

The right choice depends on the interaction type, biological question, sample constraints and desired resolution.

Quick Technology Selection Guide

Practical Planning Checklist

When planning an interaction project, you can quickly shortlist technologies by asking:

• Do I care about protein–DNA, protein–RNA, DNA–DNA or protein–protein contacts?
• How many cells or how much tissue can I realistically obtain?
• Do I need base-pair resolution, or are broader regions enough for my question?
• Is this a discovery screen, or a follow-up mechanistic study?
• Which orthogonal assays (for example Co-IP, ChIP-qPCR, reporter assays) will validate key hits?

Thinking through these points helps match your study design to the most suitable platform.

How Can Multi-Omics Designs Turn Maps into Mechanisms

Interaction maps become most powerful when combined with other omics layers.

Multi-omics designs help shift from "where things bind" to "what they do".

Overview of multi-omics study design integrating genomic, epigenomic, transcriptomic, proteomic and metabolomic layers to investigate disease mechanisms. (Hasin Y. et al. (2017) Genome Biology)

Common combinations include:

• ChIP-seq or CUT&Tag + RNA-seq
  • Link transcription factor or histone binding with gene expression changes.
• Hi-C + ChIP-seq + RNA-seq
  • Connect 3D chromatin loops, regulatory marks and transcription outcomes.
• RIP-seq or eCLIP-seq + total RNA-seq
  • Relate RBP binding patterns to splicing, stability or translation effects.
• IP-MS or RIME MS + ChIP-seq
  • Map protein complexes and their genomic occupancy in the same project.

These integrated designs are now common in epigenomic studies of development, cancer, and other complex diseases.

How CD Genomics Supports Molecular Interaction and Epigenomic Sequencing Projects

CD Genomics provides a broad portfolio of interaction and epigenomic sequencing services, including:

• ChIP-seq and CUT&Tag for protein–DNA binding maps
• RIP-seq and eCLIP-seq for protein–RNA interaction profiling
• Hi-C for 3D genome architecture

Our team supports you from experimental design and antibody selection through to bioinformatics analysis and biological interpretation.

We work with cell lines, model organisms and human research samples.

All services are provided for research use only, not for diagnostic or clinical applications.

If you are planning a molecular interaction or epigenomic sequencing project and are unsure which combination of ChIP-seq, CUT&Tag, Hi-C, RIP-seq, eCLIP-seq or IP-MS fits your question, our technical team can help.

Contact CD Genomics to discuss your study design, obtain a customized quotation, or share your project outline for a free technical evaluation.

Frequently Asked Questions About Molecular Interaction Technologies

Q1. What is the difference between ChIP-seq and CUT&Tag?

ChIP-seq and CUT&Tag both map protein–DNA binding sites across the genome, but they differ in workflow and sample requirements. ChIP-seq relies on bulk chromatin fragmentation and immunoprecipitation, while CUT&Tag uses an antibody-guided Tn5 transposase to cut and tag DNA only near the target protein, making it more suitable for low-input and precious research samples.

Q2. When should I choose RIP-seq instead of eCLIP-seq?

RIP-seq is ideal as a first screen to identify which RNAs associate with a given RNA-binding protein. eCLIP-seq is recommended when you also need nucleotide-level binding positions, or when sample amount is limited and you want a more refined interaction map.

Q3. How much material do I need for Hi-C experiments?

Standard Hi-C protocols usually require millions of cells to produce a robust genome-wide contact map, especially when high resolution is needed. Low-input and Individual-cell 3D genome approaches exist, but they use specialized protocols and involve more complex data analysis.

Q4. What is the advantage of IP-MS or RIME MS over yeast two-hybrid?

IP-MS and RIME MS capture protein–protein interactions in a cellular or nuclear context, reflecting more physiological complexes. Yeast two-hybrid screens large libraries in yeast and is powerful for discovery, but often generates more false positives that require follow-up validation.

Q5. Can CD Genomics help design a multi-omics interaction study?

Yes. CD Genomics provides project consulting to combine interaction assays such as ChIP-seq, CUT&Tag, Hi-C, RIP-seq or RIME MS with RNA-seq and other omics layers, from experimental design and antibody choice to bioinformatics analysis and interpretation.

References

1. Hasin, Y., Seldin, M., Lusis, A. Multi-omics approaches to disease. Genome Biology 18, 83 (2017).
2. Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature Communications 10, 1930 (2019).
3. Van Nostrand, E.L., Pratt, G.A., Shishkin, A.A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nature Methods 13, 508–514 (2016).
4. Sahadevan, S., Sekaran, T., Ashaf, N. et al. htseq-clip: a toolset for the preprocessing of eCLIP/iCLIP datasets. Bioinformatics 39, btac747 (2023).
5. Lieberman-Aiden, E., van Berkum, N.L., Williams, L. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
6. Sikdar, S., Datta, S., Zhang, L. et al. MHiC, an integrated user-friendly tool for the identification and visualization of significant interactions from Hi-C data. BMC Genomics 21, 256 (2020).
7. Mohammed, H., Taylor, C., Brown, G.D. et al. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nature Protocols 11, 316–326 (2016).