HELIOS NAD-Seq: A Next-Generation Capture and Sequencing Protocol for NAD-Capped RNAs with Superior Targeting and Processing

Background Nicotinamide adenine dinucleotide (NAD) can function as a non-canonical initiating nucleotide, producing NAD-capped RNAs across all domains of life. This 5′-modification has been implicated in RNA stability and host–pathogen interactions. Existing identification methods, such as NAD captureSeq, require large RNA inputs, show limited specificity, and have low sample throughput, restricting their utility for low-input or large-scale studies. Results We developed HELIOS NAD-Seq (High-efficiency Enzyme modification with Low Input Of Sample for NAD-RNA Sequencing), a protocol that combines a pyridyl-based ADP-ribosyl cyclase substrate, 3-picolylamine biotin, with early sample barcoding to enable high-yield, one-step biotinylation of NAD-RNAs. HELIOS NAD-Seq reduces RNA input requirements by at least 50-fold compared to NAD captureSeq, increases specificity for NAD-caps, and allows simultaneous preparation of libraries from at least 16 sample groups in quadruplicate within four days. Applied to Escherichia coli , HELIOS NAD-Seq confirmed NAD-capping for 89.5% of previously identified transcripts from NAD captureSeq and detected 242 additional, predominantly low-abundance RNAs. Time-course profiling of NAD-capping across the bacterial growth curve identified a core set of metabolism-related transcripts showing low expression in stationary phase, sharp peaks in early exponential growth, and decreases in the plateau phase, suggesting a link between metabolic state and NAD-capping dynamics. Conclusions HELIOS NAD-Seq is a robust and sensitive high-throughput platform for NAD-RNA profiling enabling low-input and large-scale studies. Its application to E. coli demonstrates technical advantages over existing protocols and reveals dynamic NAD-capping patterns associated with metabolism.