Next-Generation ARTR-seq: Higher-Specificity Profiling Of RNA-Protein Interactions From Low-Input Samples
SUMMARY
A refined, antibody-guided in situ reverse-transcription sequencing method that maps RNA-binding protein binding sites with reduced background signal, building on the published ARTR-seq platform
The Unmet Need: Existing RNA-binding assays struggle to capture interactions with sensitivity and low background
- RNA-binding proteins (RBPs) control how RNA is processed, stabilized, transported and translated, and more than 2,500 are predicted in humans. Their dysfunction is linked to human disease, and mapping where each protein binds is foundational for understanding gene regulation. The same information is increasingly valuable for RNA-targeted drug discovery, where pharmaceutical investment has grown rapidly. Methods that capture both stable and transient RBP-RNA interactions from small samples have been lacking.
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ARTR-seq tethers a reverse transcriptase to bound proteins through antibodies in fixed cells, copies nearby RNA in place, and maps RBP binding sites from as few as 20 cells or a tissue section, resolving changes on a 10-minute timescale. As with other in situ proximity methods, background and indirect signal affect data quality, so improving specificity is a central goal for the next generation of the approach.
The Proposed Solution: An engineered variant of ARTR-seq that lowers background signal while retaining in situ, antibody-guided capture of stable and transient RBP-RNA interactions in small samples
- The faculty inventor developed Split ARTR-Seq building on ARTR-Seq by dividing reverse transcriptase into N- and C-terminal halves that have low activity when apart and higher activity when brought into proximity or induced to assemble by a molecular cue. Antibody-guided, in-situ reverse transcription profiles RNA bound by target proteins, while the split-enzyme design is intended to reduce background; a later modification adds a nanobody to improve signal-to-noise. The method avoids UV cross-linking and immunoprecipitation and can be applied to RNA interactions involving two proteins.
ADVANTAGES
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No UV crosslinking or immunoprecipitation
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Maintains low-input capability
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Improved signal-to-noise with nanobody modification
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Reduced reverse-transcription background
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Capture of transient RNA–protein interactions
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Short-interval interaction profiling
APPLICATIONS
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RNA-targeted drug discovery: mechanism of action and target engagement
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Profiling of nascent RNA processing by RNA-binding proteins
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Off-target RBP binding analysis for RNA therapeutics
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Low-input and tissue-section profiling of RBP-RNA interactions
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Time-resolved studies of dynamic RNA-protein binding