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Probe Design

Padlock probe design is the first step of the PRISM / SPRINTseq workflow. Each probe is a linear oligonucleotide (~70--100 nt) consisting of two target-complementary arms flanking a backbone sequence that encodes the gene barcode.

Design Pipeline

The Huang Lab maintains an open-source probe design toolkit:

Resource Link Description
probe-designer (Python) GitHub: HuangLab-PKU/probe-designer Core design pipeline: target selection, arm design, specificity filtering, barcode assignment
Probe Design Web App (internal deployment) Interactive web UI for designing and managing probe panels (FastAPI + Vue 3)

Probe Architecture

5'─[Left Arm]─[Backbone / Barcode]─[Right Arm]─3'
     ~~20 nt        ~~30-60 nt          ~~20 nt
       │                                   │
       └──── Hybridize to target RNA ──────┘
                (adjacent binding)
  • Arms: Complementary to the target mRNA, designed to bind adjacent positions. Tm matching is critical.
  • Backbone: Contains the gene-identifying barcode sequence, detection probe binding sites (PRISM) or sequencing primer binding sites (SPRINTseq), and the RCA primer binding site.
  • Specificity: Arms are checked against the transcriptome for off-target binding. Only probes passing specificity filters are included in the final panel.

Design Considerations

  • Target selection: Choose exonic regions with low secondary structure and no SNPs in the arm-binding region.
  • Tm balancing: Both arms should have similar melting temperatures (~55--60°C at hybridization salt conditions).
  • Barcode assignment: For PRISM, barcodes map to color-intensity codes. For SPRINTseq, barcodes are read by sequencing.
  • Panel size: Typically 30--100+ genes per panel; limited by barcode space (PRISM) or sequencing cycles (SPRINTseq).
  • Synthesis: Order as standard oligos from IDT or similar vendors. Pool at equimolar concentrations.

iLock Probe Variant

iLock (invader padLock) probes add a 5' non-complementary flap to the standard padlock design. Before ligation, Taq DNA Polymerase cleaves this flap via its 5'→3' flap endonuclease activity, providing an additional specificity checkpoint. See the iLock publication.

Unpublished

iLock probe design parameters are currently internal to the lab.