RADAR: RNA Accessibility profiling via Deaminase-Annotated Reactivity
SUMMARY
Higher-sensitivity RNA accessibility and protein-binding profiling with substantially lower sequencing demand is provided by RADAR, an enzymatic probing platform that uses evolved deaminases to mark accessible nucleotides in vitro and in cells
The Unmet Need: RNA accessibility methods need greater sensitivity with substantially less sequencing
- Widely used chemical probing approaches can produce signals that impede reverse transcription and require substantial sequencing to achieve useful coverage. These limitations make large-scale profiling of RNA structure and protein interactions more demanding.
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Advances in high-throughput sequencing and engineered enzymes are creating opportunities for scalable, multiplexed RNA analysis. Interest in mapping RNA structure alongside RNA-binding protein engagement is also increasing, with methods that work in vitro and in cells offering a route to broader functional profiling.
The Proposed Solution: Engineered deaminases mark accessible RNA nucleotides with sequencing-compatible mutation signatures
- The faculty inventor developed RADAR (RNA Accessibility profiling via Deaminase-Annotated Reactivity)- a technique using evolved hyperactive deaminases to modify accessible adenosines, which are detected as A-to-G changes after reverse transcription; engineered cytosine deaminases can similarly generate C-to-U signatures. The method operates under mild conditions, preserves RNA integrity, and produces dense reactivity profiles compatible with sequencing, distinguishing it from chemical probing methods that can impede reverse transcription.
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Reported validation includes reproducible measurements across a pooled set of 240 RNAs with known structures, detection of structure- and protein-associated accessibility changes in vitro and in cells, and stronger signal-to-noise with comparable coverage. RADAR can support profiling of RNA folding and protein engagement, including coexisting conformations and ribosome occupancy.
ADVANTAGES
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Higher signal-to-noise RNA profiling
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Reduced sequencing-read requirements
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Reverse-transcription-compatible mutation signatures
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Preserved RNA integrity
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In vitro and cellular measurements
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Combined structure and protein-binding analysis
APPLICATIONS
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RNA structure and accessibility mapping
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RNA-binding protein interaction profiling
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Ribosome occupancy analysis in bacterial transcripts