Design Experiments From Reported Results

 Long Analysis Plan Copied

Evidence Base: Dosa Covalent RNA Barcoding

The Dosa method fuses a protein of interest to catalytically inactive E. coli TrmA(E358Q) plus a separate 19-nt stem-loop RNA barcode (10-nt barcode region); covalent protein–RNA linkage forms in vivo, and subsequent enrichment, reverse transcription, and Illumina sequencing directly map barcode → variant → biochemical phenotype without purifying each protein Dosa covalent barcoding preprint.

All five correlations are from the Dosa paper itself (n≈2–3 replicates each); the solubility assay is the noisiest (r=0.81), a key caveat for planning.

Bioinformatics/Coding Analysis Plan

• Barcode–variant association: Process PacBio long reads (bcl2fastq → PANDASeq assembly → per-molecule barcode/variant pairing); collapse to barcode–genotype dictionary with redundancy checks; target median ~10 barcodes/variant as in the FLAG library (152 variants, ~1,900 barcodes) Dosa library sizing.
• Enrichment scoring: Compute log2(barcodes on capture beads / barcodes on Ni-NTA input) per variant; statistical testing via prop.test/statsmodels; replicate correlation QC (target r ≥ 0.9) Dosa enrichment metric and data access.
• Assay-specific scoring: Protease cleavage (barcode depletion = cleavable); solubility as log2(supernatant/pellet) after 8 M urea fractionation; Aβ(1-42) 794-variant library validated positions 17–20 and 34–36 as solubility-increasing, consistent with type I filament structural context.
• Bridge to harsh in vitro conditions: Because the barcode is covalently attached, enriched complexes survive stringent washes/denaturants — plan tolerance tests for 8 M urea, high temperature, and detergents; verify no barcode leakage via stem-loop excess during lysis (authors' mitigation) Dosa limitations.

Context from TrmA Biology

TrmA installs m5U54 on tRNA; trmAΔ knockouts lose m5U54 and show modification crosstalk (acp3U47 decreases, s4U increases in tRNAPhe), confirming the enzyme's known target and supporting its engineered E358Q derivative as a dead-transferase that still binds RNA stem-loops covalently TrmA m5U54 crosstalk evidence. TrmA also appears in activity-based RNABPP-PS profiling of bacterial RNA-modifying enzymes, with in vitro crosslinking of purified TrmA to RNA demonstrated RNABPP-PS TrmA crosslinking.

Known unknowns: Whether the E358Q fusion context systematically biases folding-sensitive assays (the paper acknowledges this); iterative selection is not possible because barcodes are separate from mRNA. Data: SRA PRJNA1329341; PRIDE PXD068701 for tRNA-MS.