FAST-MaP (Fast and Accessible Sequencing Technology for Mutational Profiling) is a laboratory protocol for per-nucleotide characterization of RNA structure that is designed to be accessible to researchers without sequencing infrastructure or bioinformatics expertise. The method uses orthogonal chemical probes (reported as 2A3 and DMS) to modify RNA, followed by reverse transcription and PCR to generate double-stranded DNA amplicons that are submitted to a commercial primer-less sequencing service. FASTQ output from the service is processed through a freely accessible web server to yield normalized nucleotide reactivity profiles within minutes. The complete protocol, from DNA template to structural data, can be completed in approximately one week. The authors illustrate the workflow on a 659-nucleotide RNA, showing how to evaluate buffer-dependent structure preservation and to test computational or cryo-EM-derived secondary and tertiary structure models.
FAST-MaP is presented as an end-to-end experimental pipeline for mapping RNA chemical reactivity at single-nucleotide resolution using widely available molecular-biology techniques paired with an external sequencing provider and an online analysis tool. Key conceptual stages are: chemical probing of RNA with orthogonal reagents, reverse transcription to encode modifications as mutations, PCR amplification to make sequencing-ready dsDNA amplicons, submission of amplicons to a commercial primer-less sequencing service, and automated processing of returned FASTQ files on a public web server to produce normalized reactivity profiles. The workflow emphasizes ease of use and minimal requirement for local sequencing or computational infrastructure.
The protocol uses orthogonal chemical probes to mark nucleotides according to their structural accessibility. The authors report use of 2A3 and DMS as the orthogonal reagents in their demonstrations. Chemical modification is the initial experimental step and is central to converting structural information into sequence-detectable signals after reverse transcription. Specific reaction conditions, concentrations, times, and buffer details were described in the authors’ full protocol and supplementary materials; this summary reflects only that orthogonal probes are used to obtain complementary reactivity information.
After chemical modification, RNA is reverse-transcribed in conditions that convert modification sites into mutations or stops that can be read out by sequencing. The resulting cDNA is PCR-amplified to generate double-stranded DNA amplicons suitable for standard sequencing. Instead of performing sequencing in-house, the protocol submits these PCR products to a commercial primer-less sequencing service. The strategy of primer-less submission is a central feature enabling laboratories without sequencing platforms or library-preparation expertise to obtain raw sequencing reads (FASTQ) for downstream analysis.
Returned FASTQ files are uploaded to a freely accessible web server provided by the authors’ group. The server processes the sequencing reads and produces normalized nucleotide reactivity profiles within minutes. These normalized reactivity profiles enable per-nucleotide interpretation of chemical probing results and can be used to compare experimental conditions or to test structural models. The availability of a public processing pipeline is intended to remove the need for users to develop custom bioinformatics workflows.
The authors demonstrate FAST-MaP on a 659-nucleotide RNA. In that demonstration, they show how the workflow can be used to test whether an RNA’s structure is preserved across different buffer conditions, and how the experimental reactivity data can be used to evaluate specific secondary and tertiary structure predictions derived from computational modeling or cryo-electron microscopy. Detailed examples, figures, and raw data for these demonstrations are provided in the preprint and supplementary materials linked by the authors.
According to the authors, the complete FAST-MaP protocol—from DNA template to processed structural reactivity data—can be completed in approximately one week. The protocol is framed as requiring only standard molecular-biology skills and routine laboratory equipment; it is specifically intended to lower technical barriers by avoiding the need for local sequencing platforms or bioinformatics expertise. The public web server and primer-less sequencing submission together aim to make chemical mapping studies more accessible to a broader group of laboratories working on RNA structure and function.
The authors report that J.V., H.M.B., and R.D. filed an invention disclosure with Stanford University covering the FAST-MaP protocol and associated analysis tools; no other competing interests were declared. Funding sources declared include the National Institute of General Medical Sciences (NIGMS) R35 GM122579 and the Howard Hughes Medical Institute. The preprint provides links to supplementary material and to data/code resources maintained by the authors’ group. For correspondence the preprint lists Rhiju Das (rhiju@stanford.edu). The protocol and analysis tools are released alongside the preprint to enable adoption, and the authors note that the server produces normalized reactivity profiles within minutes of FASTQ upload.
The preprint and supplementary materials include detailed protocol steps, example datasets, and links to the public web server and associated resources. Users interested in applying FAST-MaP should consult those materials for exact reagent lists, experimental conditions, and instructions for sequencing submission and web-server usage. As this work is presented as a preprint, it has not been certified by peer review; readers should consult the original preprint for full experimental detail and any subsequent peer-reviewed publication.