Technologies Overview¶

Both PRISM and SPRINTseq are padlock probe-based spatial transcriptomics technologies. They share a common wet-lab workflow for library construction, but diverge in how the spatial signal is read out.

Shared Workflow¶

graph LR
    P["<b>Probe Design</b><br/><small>Padlock probes</small>"] --> A
    A["<b>1. Sample Prep</b><br/><small>FF or FFPE</small>"] --> B["<b>2. Hybridization</b><br/><small>Probes bind RNA</small>"]
    B --> C["<b>3. Ligation</b><br/><small>Circularization</small>"]
    C --> D["<b>4. RCA</b><br/><small>Rolonies</small>"]
    D --> E1["<b>PRISM</b><br/><small>Single-round imaging</small>"]
    D --> E2["<b>SPRINTseq</b><br/><small>In-situ sequencing</small>"]

    style P fill:#F5ECDA,stroke:#B5832F,stroke-width:2px
    style E1 fill:#E9EEE6,stroke:#5F7A57,stroke-width:2px
    style E2 fill:#E7EDF1,stroke:#5B7488,stroke-width:2px

Step-by-Step¶

Probe Design¶

Target-specific padlock probes are designed computationally. Each probe is a linear oligonucleotide with two target-complementary arms flanking a unique backbone barcode sequence. See Probe Design.

Hybridization & Ligation¶

Padlock probes hybridize to their RNA targets in fixed tissue. When both arms bind adjacently, the probe is ligated into a circle by SplintR ligase (PBCV-1 DNA Ligase), which works on DNA:RNA hybrids. Only perfectly matched probes are circularized, providing single-nucleotide specificity.

Rolling Circle Amplification (RCA)¶

Phi29 DNA Polymerase continuously copies the circularized probe, producing a long single-stranded DNA concatemer called a rolony (rolling-circle colony). Each rolony contains thousands of tandem copies of the probe sequence. See RCA Mechanism.

Readout¶

PRISMSPRINTseq

Fluorescent detection probes carrying color-intensity barcodes hybridize to rolonies. Each gene is assigned a unique combination of fluorescence color and intensity level. A single round of multi-channel imaging captures all signals, and a radius vector decoding algorithm identifies each gene.

Key advantages: Fast (one imaging round), simple optics, compatible with conventional microscopes. Up to 64-plex.

PRISM Details

Rolonies undergo multi-cycle sequencing-by-synthesis (SBS) directly on tissue. SPRINTseq uses hybrid block coding and signal dilution to overcome crowdedness in dense tissues.

Key advantages: Higher multiplexing capacity, variant/isoform detection, near optical diffraction-limit resolution. 108-gene panel profiled in < 9.5 h.

SPRINTseq Details

Choosing Between PRISM and SPRINTseq¶

	PRISM	SPRINTseq
Best for	Rapid profiling of defined gene panels	Deep targeted transcriptome with variant info
Readout	Single-round fluorescence imaging	Multi-cycle in-situ sequencing
Encoding	Color-intensity barcodes (radius vector)	Hybrid block coding
Imaging rounds	1	Multiple (sequencing cycles)
Throughput	Very fast	< 9.5 h per brain slice
Equipment	Conventional fluorescence microscope	Fluorescence microscope + fluidics
Multiplexing	Up to 64-plex	Scales with sequencing cycles

Key Publications¶

Technology	Publication	Journal	DOI
PRISM	High-plex spatial RNA imaging in one round with conventional microscopes using color-intensity barcodes	Nature Biotechnology (2025)	10.1038/s41587-025-02883-7
SPRINTseq	Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding	PNAS 120(47) (2023)	10.1073/pnas.2309227120
iLock probes	Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection	RNA 25(1) (2019)	10.1261/rna.066753.118

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