What is rG4-RIP mRNA sequencing, and how does it work?

rG4-RIP mRNA sequencing combines RNA immunoprecipitation (RIP) of G-quadruplex (G4)-rich RNAs or their binding proteins with high-throughput sequencing to identify transcripts associated with RNA G-quadruplexes (rG4s) and RNA-binding proteins (RBPs) across the transcriptome.

How is rG4-RIP different from standard RIP-Seq or rG4-Seq?

Unlike standard RIP-Seq, which captures RBP-bound RNAs in general, rG4-RIP specifically targets transcripts associated with rG4 structures and their interacting proteins; rG4-Seq, meanwhile, maps the physical location of G4 structures but does not measure binding enrichment of RBPs.

What kinds of research questions can rG4-RIP mRNA Seq address?

rG4-RIP can uncover which mRNAs harbour folded G4 structures and bind specific RBPs, reveal their networks under stress or disease conditions, and support target discovery for RNA-structure mediated regulation or biomarker development.

What sample input is required for rG4-RIP mRNA Seq, and are there special considerations?

RNA or RNA–protein lysates with sufficient quantity and quality (e.g., RIN ≥ 8, minimal degradation) are required; special considerations include including input controls and optional ionic-condition comparisons (e.g., K⁺ vs Li⁺) to validate G4-dependence of binding.

How is the data from rG4-RIP mRNA Seq interpreted, and what form of results will I get?

The analysis pipeline will produce enrichment tables of transcripts (IP vs input), annotation of G4 motifs, RNA–protein interaction networks, functional pathway enrichment, and visualisation (volcano plots, heatmaps, network maps) to translate raw reads into meaningful biological insights.

Can rG4-RIP mRNA Seq be used to support drug discovery or biomarker development?

Yes. By identifying transcripts and RBPs associated with rG4 structures under disease-related conditions, rG4-RIP mRNA Seq enables the prioritisation of novel therapeutic targets or biomarkers linked to RNA structural regulation.

What are the limitations or things to watch out for with rG4-RIP mRNA Seq?

Key considerations include antibody specificity for rG4s or RBPs, fold-change thresholds for enrichment, possible indirect binding artefacts, and the need to validate findings by complementary methods (such as structure mapping, functional assays).

How long does the workflow take, and what support is provided by a CRO?

While timelines vary by project complexity, the service provider should offer design consultation, sample QC, sequencing, bioinformatics reporting, and publication-ready outputs with strong CRA (Conversion-Ready Answers) support.

Can rG4-RIP mRNA Seq be combined with RNA-structure mapping methods like SHAPE-Seq or DMS rG4-Seq for validation?

Yes, these combined approaches confirm structural dependence and interaction specificity.

rG4-RIP mRNA Seq Service | RNA G-Quadruplex & RBP Interaction Mapping

Step	Description
Raw Data QC	Validate sequencing quality (base scores, adapter contamination, duplicate reads) to ensure data integrity.
Read Alignment & Quantification	Map reads to the reference genome/transcriptome and compute transcript abundance (IP and input libraries).
Differential Enrichment (IP vs Input)	Identify transcripts significantly enriched in the IP fraction compared to input, signalling rG4-/RBP-association.
rG4 Motif Annotation & Network Mapping	Annotate transcripts for G-quadruplex (G4) motif status and construct RNA–protein interaction networks linking rG4s and RBPs.
Functional Enrichment & Pathway Analysis	Perform GO/KEGG/GSEA to interpret biological context of enriched transcripts and identify regulatory pathways.
Optional Comparative Condition (K⁺ vs Li⁺)	For mechanistic studies, compare enrichment under K⁺ (G4-stable) and Li⁺ (G4-unstable) conditions to validate rG4-dependence.
Visualisation & Reporting	Generate figures such as volcano plots, heatmaps, network graphs, and provide executive summary of biological interpretations.

Technology	Core Purpose	Input Material	Key Outputs	Resolution / Focus	Ideal Use Case
rG4-RIP mRNA Seq	Identify mRNAs and RNA-binding proteins (RBPs) associated with RNA G-quadruplexes (G4s) via immunoprecipitation + sequencing	Total RNA or RNP lysate	Enriched transcripts, RBP-mRNA interaction networks	Transcript-level binding/enrichment with structural context	Studies of rG4-dependent RNA–protein networks, drug/biomarker research
DMS rG4-Seq	Map in vivo folded rG4 structures using dimethyl sulfate (DMS) chemical probing + RT-stop or mutation profiling	Live cells or extracted RNA treated with DMS	rG4 folding sites, RT-stop signatures, K⁺/Li⁺ dependence	Single-nucleotide structure resolution	When you need to know where rG4s fold in transcripts and confirm structure in cell context
rG4-Seq	Profile transcriptome-wide potential rG4-forming sequences (in vitro or ex vivo) using RT-stall or other read-through outcomes	Purified poly(A) RNA, usually high input	Global map of candidate rG4 sites, structure potential	Single-nucleotide or motif resolution (in vitro)	When you aim to catalogue possible rG4 motifs or generate structural hypotheses
Standard RIP-Seq	Capture RNAs bound by a specific RBP without explicit structural focus	RNA from RBP immunoprecipitate	List of RBP-associated RNAs with fold-enrichment	Transcript-level binding	Broad RBP interaction profiling regardless of G-quadruplex involvement

Parameter	Specification
Input RNA Quantity	≥ 1–2 µg total RNA or equivalent RNP complex
RNA Concentration	≥ 50 ng/µL, OD260/280 ratio ~1.8–2.0 for purity
Library Sequencing	Illumina PE150 (paired-end 150 bp reads)
Sequencing Depth	Standard: ~6 Gb per library
Controls	Input (total RNA) required; IgG‐control optional
Recommended Replicates	≥2–3 biological replicates (IP + input per condition)
Optional Condition	Parallel K⁺ vs Li⁺ treatment to assess rG4-dependence

This study focuses on RNA G-quadruplexes (rG4s) — guanine-rich RNA sequences that fold into four-stranded structures. Although rG4-forming sequences are abundant in transcriptomes, their physiological roles in the central nervous system were largely unknown. The researchers identified the RNA-binding protein DNAPTP6 in mouse forebrain as having a strong affinity for rG4s and hypothesized that DNAPTP6 might coordinate stress granule (SG) assembly via rG4-dependent mechanisms.

Proteomic screening of mouse forebrain lysates using an rG4-specific antibody (BG4) to pull down rG4-associated proteins, followed by LC-MS/MS to identify DNAPTP6.
Biochemical assays: Electrophoresis mobility shift assays (EMSA) and surface plasmon resonance (SPR) to measure the binding affinity of DNAPTP6 for rG4 versus non-rG4 RNA.
Phase separation assays in vitro: testing whether DNAPTP6 undergoes liquid-liquid phase separation (LLPS) and whether addition of rG4 RNA enhances droplet formation.
Cellular experiments in neurons (mouse cortical neurons & Neuro-2A cells): Knockdown of DNAPTP6, arsenite-induced oxidative stress to trigger SGs, immunostaining for SG markers (e.g., G3BP1), and functional readouts including spontaneous excitatory postsynaptic currents (sEPSCs) and cleaved caspase-3 immunoreactivity (a marker of neuronal cell death).

DNAPTP6 binds rG4s with high selectivity compared with non-rG4 sequences, via its CCD and IDR2 domains.
In vitro, DNAPTP6 forms spherical droplets (LLPS), and these droplets grow in size when rG4 oligomers or rG4-containing mRNAs (e.g., Mark2 3′UTR) are added, but not when non-rG4 mutants are used.
In neurons, DNAPTP6 colocalises with SG marker G3BP1 under stress. Knockdown of DNAPTP6 reduces SG formation under sodium arsenite stress, reduces sEPSC frequency (indicating synaptic dysfunction), and increases neuronal apoptosis (cleaved caspase-3) under stress conditions.
The mRNAs containing multiple rG4-forming sequences are significantly enriched in SGs; rG4 motifs—particularly in CDS and 3′UTR—correlate with higher SG recruitment.

DNAPTP6 colocalises with stress granule marker G3BP1 and recruits rG4-containing mRNA into stress granules under oxidative stress. Confocal micrographs showing colocalisation of DNAPTP6 and G3BP1 in stress granules of Neuro-2A cells treated with sodium arsenite (NaAsO₂). Mark2 rG4-containing mRNA (fluorescent-labelled) is recruited into DNAPTP6-positive SGs, whereas 7-deazaG (G4-mutant) transcript shows no recruitment.

The study demonstrates that rG4s act not just as static motifs, but actively recruit mRNAs into stress granules through binding by DNAPTP6. The rG4-dependent phase separation of DNAPTP6 drives SG assembly, which in turn is crucial for neuronal resilience under oxidative stress. Disruption of this mechanism (via DNAPTP6 knockdown) impairs SG formation, leads to synaptic dysfunction, and neuronal cell death. Thus, rG4-mediated RNA–protein phase separation emerges as a key mechanism in neuronal homeostasis and stress-response biology.