Library Construction Strategies

Padlock probe-based spatial transcriptomics can use different ligation strategies depending on the substrate (RNA or cDNA) and probe design. The choice affects detection efficiency, specificity (SNP discrimination), and self-ligation risk.

Strategy Comparison

Strategy	Substrate	Ligase	Detection Efficiency	SNP Specificity	Self-Ligation Risk	Extra Steps
Direct RNA (dRNA)	mRNA	SplintR (PBCV-1)	Highest	None	High	None
Chimeric Padlock dRNA	mRNA	T4 RNA Ligase 2	Higher than dRNA	None	High	None
cDNA	cDNA (after RT)	T4 DNA Ligase / Tth	~⅕ of dRNA	Yes	Low--moderate	Reverse transcription + RNase H
iLock dRNA	mRNA	SplintR (after Taq activation)	~1/10 of dRNA	Yes	Minimal	Taq activation step

Direct RNA (dRNA)

The simplest approach. Standard DNA padlock probes hybridize directly to mRNA, and SplintR ligase (PBCV-1 DNA Ligase) ligates the DNA nick on the RNA template (DNA:RNA hybrid).

Pros:

• Highest detection efficiency (no conversion loss)
• Fewest steps (no reverse transcription)

Cons:

• Almost no single-nucleotide specificity -- SplintR ligase tolerates mismatches at the ligation junction
• Self-ligation risk: SplintR ligase can ligate probes that circularize without a template (template-independent self-ligation), producing false positives

Protocol: RCA: Direct RNA | Used in: Standard PRISM and SPRINTseq protocols (current default).

Chimeric Padlock Direct RNA

A variant of direct RNA where the 3' and/or 5' terminal nucleotides of the padlock probe are replaced with ribonucleotides (chimeric DNA-RNA probe). This creates an RNA-RNA hybrid at the ligation junction instead of a DNA-RNA hybrid.

Ligase: T4 RNA Ligase 2 (T4Rnl2), which ligates RNA-RNA hybrids more efficiently than SplintR ligates DNA-RNA hybrids. SplintR can also be used but is less efficient on chimeric substrates.

Pros:

• Higher detection efficiency than standard dRNA (more stable RNA-RNA junction)
• No extra steps compared to standard dRNA

Cons:

• Same lack of SNP specificity as standard dRNA
• Same self-ligation risk
• Chimeric oligo synthesis is more expensive

Reference: Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection. RNA 25(1), 82--89 (2019). 10.1261/rna.066753.118

cDNA

Reverse transcription converts mRNA to cDNA first. Padlock probes then hybridize to the cDNA strand, and a DNA-templated ligase (T4 DNA Ligase, Tth Ligase, etc.) ligates the nick.

Pros:

• SNP specificity: DNA-templated ligases discriminate single-nucleotide mismatches at the ligation junction
• Lower self-ligation risk than SplintR (though template-dependent self-ligation between probes is still possible -- use blocking oligos to mitigate)

Cons:

• Detection efficiency ~⅕ of direct RNA (conversion loss during reverse transcription)
• Extra step: reverse transcription
• Extra step: RNase H digestion to remove the RNA strand after RT, exposing the cDNA for padlock hybridization

Protocol: RCA: cDNA — includes reverse transcription, RNase H digestion, and T4 DNA Ligase ligation steps.

iLock Direct RNA

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. This structure-specific cleavage requires correct base-pairing at the cleavage site, providing a dual specificity checkpoint (cleavage + ligation).

Pros:

• Highest specificity: SNP discrimination at the flap cleavage site + ligation junction
• Minimal self-ligation: The flap must be cleaved before ligation can occur, fundamentally preventing template-independent circularization

Cons:

• Lowest detection efficiency (~1/10 of dRNA): Taq activation requires elevated temperature (~45--51°C) where the short flap duplex is unstable, leading to low activation efficiency
• Extra step: Taq activation (45°C, 60 min)

Potential optimization

Increasing Taq polymerase concentration + 37°C overnight activation may improve activation efficiency. This is an active area of optimization.

Unpublished

iLock probe protocols are currently internal to the Huang Lab.

Reference: Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection. RNA 25(1), 82--89 (2019). 10.1261/rna.066753.118

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Choosing a Strategy

graph TD
    Q1{"Need SNP<br/>specificity?"} -->|No| Q2{"Maximize<br/>detection?"}
    Q1 -->|Yes| Q3{"Minimize<br/>false positives?"}
    Q2 -->|"Yes, max efficiency"| A["Chimeric Padlock dRNA"]
    Q2 -->|"Standard is fine"| B["Direct RNA (dRNA)"]
    Q3 -->|"Specificity > sensitivity"| C["iLock dRNA"]
    Q3 -->|"Balance both"| D["cDNA"]

    style A fill:#E9EEE6,stroke:#5F7A57
    style B fill:#E9EEE6,stroke:#5F7A57
    style C fill:#F5ECDA,stroke:#B5832F
    style D fill:#E7EDF1,stroke:#5B7488

For most spatial transcriptomics applications where detection efficiency is the priority and single-nucleotide specificity is not required, direct RNA is the recommended default strategy.