HOST-DIALOGIntegrated Functional Assay Suite
Visual Protocol Overview · for benchside reference
SOP-HD-001 · v1.0
April 2026 · DRAFT
Karlsen / Bollvåg et al.

One master culture, split four ways, sampled through three orthogonal axes — radiolabel incorporation, tRNA charging state, and small RNA profile — converging onto a single mechanistic readout of GatCAB-mediated tRNA-Asn maturation in S. aureus.

01 Timeline · split-culture, drug application, sampling all times relative to drug addition (T = 0)
T−90preculture dilute T−15label flasks T 0SPLIT + drug T 15 T 30³H pulse on T 45quench + TCA T 60 T 120 growth phase: OD₆₀₀ 0.05 → 0.5 MASTER 100mL vehicle DMSO ESYLWS 0.5× MIC ESYLWS 1× MIC CAM control 15 min ³H pulse ● clean-arm RNA samples ▮ hot-arm pulse window Day 0 · single-day execution CDM, 37 °C, 200 rpm — strain: USA300 JE2
02 Three orthogonal readouts · downstream workflow samples at T+45 (hot) and T+15/30/45/60/120 (clean)
ARM A · HOT ARM B · ACID-UREA ARM C · SMALL RNA SEQ Translation flux per residue Direct tRNA charging state tRF & sRNA regulatory profile SOP 2 · ³H-Asp/Asn/Leu/Gly SOP 3 · low-pH PAGE northern SOP 4 · CIP+PNK + AlkB + NEBNext 1. Add ³H-amino acid at T+30 5 µCi/mL final, 4 tracers in parallel 2. 15 min pulse · ice quench 3 × 1 mL into 10 % TCA on ice 3. Filter onto GF/C · hot-TCA wash 90 °C, 20 min — strips RNA signal 4. Scintillation count (DPM) Tri-Carb · ³H or dual ³H/¹⁴C channel DELIVERABLE [³H-Asn] / [³H-Leu] ratio Per condition × replicate. GatCAB block: ratio drops < 0.6 at sub-MIC ESYLWS. 1. Acid-fix pellet · pH 4.5 0.3 M NaOAc cold — preserves aa-bond 2. Acid phenol extract · ethanol pp. Everything cold; do not heat-denature 3. 8 M urea PAGE · 100 mM NaOAc pH 5 8 V/cm, 4 °C, 4–6 h 4. Northern · ³²P-tRNA-Asn probe + tRNA-Leu specificity control DELIVERABLE 3-band charging spectrum Asp-tRNA-Asn band rises = direct GatCAB block. veh 0.5× 1× deAc ↑ Asn-tRNA-Asn ↓ uncharged 1. TRIzol + miRNeasy hybrid Retains 15–200 nt fraction 2. CIP + T4 PNK end-repair Removes 2′,3′-cyclic-P (tRF-essential) 3. AlkB demethylation Strips m1A/m3C/m1G — RT-permissive 4. NEBNext lib · 50 bp SE @ Nord U. 10 M reads/sample · MINTmap pipeline DELIVERABLE tRF-3b^Asn quantification Sense to DMBT1-mRNA window. Stringent-response sRNA cohort, global regulatory landscape. INTEGRATED DATASET Mechanistic concordance across three axes flux ⇄ charging state ⇄ regulation — single biological state, three orthogonal probes
03 Mechanistic readouts · diagnostic fingerprints expected three-axis signatures per scenario
Hypothesis confirmed

Clean GatCAB block

ESYLWS hits transamidation specifically. Asn codons stall while Asp codons stay open. The headline result.

Asn/Leu ↓
Asp-tRNA ↑↑
tRF-3b ↑
Off-target

General translation hit

Looks like chloramphenicol. All four tracers drop in proportion; no charging-state shift on the northern.

All ↓ equal
No shift
Stringent ↑
Cell-wall axis

MurG/MurF arm dominant

Self-activation hypothesis branch: Gly into peptidoglycan drops selectively, protein synthesis preserved.

Asn/Leu ≈ 1
No shift
Cell-wall sRNA
The full mechanism

Dual hit · the strongest case

GatCAB and MurG/MurF both engaged. Asp-tRNA-Asn rises, Gly-PG drops, tRF-3b^Asn enriched.

Asn/Leu ↓
Asp-tRNA ↑↑
tRF + PG sRNA
HOT ARM · radiation
ACID-UREA · clean RNA
SMALL RNA SEQ
SHARED master / split
All arms originate at T = 0 from one synchronised culture.