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CAM-seq: Chemical Cooperative Catalysis-Assisted M6A Sequencing

Lead Inventor: Chuan He

SUMMARY

More accurate, low-input mapping and quantification of RNA m⁶A modifications is achieved by CAM-seq, a chemically mild sequencing method that identifies sites at single-base resolution while reducing RNA damage and background noise

The Unmet Need: Harsh m⁶A sequencing conditions can damage RNA and compromise measurement accuracy

  • Existing nitrite-based methods may rely on strongly acidic conditions that degrade RNA, while incomplete reactions or residual protecting-group adducts can introduce background noise and reverse-transcription stops. These limitations can increase input requirements and hinder precise quantification of m⁶A sites across the transcriptome.

  • RNA modification research is moving toward transcriptome-wide, base-resolution maps that quantify modification levels rather than merely identify candidate sites. This creates an opportunity for sequencing chemistry that preserves RNA integrity while improving sensitivity, reducing background, and supporting analysis of fragmented or limited-input samples.

The Proposed Solution: Cooperative carbonyl and Lewis acid catalysis enables mild, base-resolution m⁶A sequencing

  • The faculty inventor developed CAM-seq (chemical cooperative catalysis-assisted m6A sequencing ) combining a carbonyl catalyst, a Lewis acid, and nitrite under near-neutral to mildly acidic conditions to convert ordinary adenine to inosine, which sequencing reads as a guanine-like signal, while m⁶A resists deamination and remains read as adenine. Reversible kethoxal protection of guanine, together with optimized reaction and reverse-transcription conditions, helps limit RNA damage, unwanted base conversion, and background errors. Unlike earlier nitrite-based approaches, the method is designed to operate under milder conditions.

ADVANTAGES

  • Single-base m⁶A mapping and quantification

  • Low-input RNA profiling

  • Reduced RNA degradation

  • Estimated background below 0.5%

  • Compatibility with fragmented RNA

  • Potentially lower sequencing costs

APPLICATIONS

  • Transcriptome-wide m⁶A profiling in human cells

  • RNA modification analysis from low-input samples

  • m⁶A mapping in Arabidopsis and maize

PUBLICATIONS