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 (general) — Tth / HiFi Taq for SNP	~⅕ of dRNA	Yes, but only with a high-fidelity thermostable ligase	Low--moderate	Reverse transcription + RNase H
iLock dRNA	mRNA	SplintR (after Taq activation)	~1/10 of dRNA	Yes	Minimal	Taq activation step

SNP specificity is a property of the ligase, not just the strategy

Moving to a cDNA (DNA:DNA) substrate is necessary for allele discrimination but not sufficient. Running the cDNA route with T4 DNA Ligase at 37°C -- the default in the cDNA protocol -- does not give you SNP calling. See Ligase Fidelity below.

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 is achievable -- a DNA:DNA junction is the substrate a high-fidelity DNA ligase needs. Realizing it requires the right ligase (see Ligase Fidelity); the standard protocol's T4 DNA Ligase is not enough on its own
• 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. For allele-level work, swap in a high-fidelity thermostable ligase (Tth or HiFi Taq) run at 45--65°C.

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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Ligase Fidelity (SNP Discrimination)

Choosing the strategy is only half the decision. Once the substrate is DNA:DNA, which ligase you use, and at what temperature, decides whether single-nucleotide variants are callable at all.

A systematic benchmark tested five DNA ligases against all 256 combinations of bases at the ligation junction (16 splint NN pairs x 4 upstream 3' bases x 4 downstream 5' bases), scoring how often each enzyme ligated a substrate it should have rejected.¹ Thermophilic ligases ran at 55°C, mesophilic ones at 37°C, 30 min, 50 RPLU each.

Mis-ligations at >50% yield (lower is better; 240 of the 256 combinations carry at least one mismatch):

Ligase	Class / cofactor	Assay temp	Single-mismatch	...on the 3'-OH side	...on the 5'-P side	Double-mismatch
Tth DNA Ligase	Thermophilic, NAD⁺	55°C	7	0	7	0
HiFi Taq DNA Ligase (NEB M0647)	Thermophilic, NAD⁺	55°C	21	3	18	not reported
Ampligase	Thermophilic, NAD⁺	55°C	37	5	32	not reported
E. coli DNA Ligase	Mesophilic, NAD⁺	37°C	73	25	48	4
T4 DNA Ligase	Mesophilic, ATP	37°C	78	30	48	17

What this means in practice

For ordinary panels, T4 (and SplintR) are fine

When the specificity element is arm hybridization -- the normal case for a targeted gene panel -- T4 DNA Ligase on the cDNA route and SplintR on the direct-RNA route are entirely adequate, and they are the defaults for good reason: they are efficient, work at 37°C, and need no thermostable-ligase logistics. A mismatch tolerated at the junction only matters if you were trying to read that base.

For SNP-level resolution, T4 and Ampligase are not sufficient

T4 DNA Ligase mis-ligated 78 single-mismatch substrates -- and still ligated 17 substrates carrying two mismatches. It cannot distinguish alleles. Ampligase, despite being thermostable, mis-ligated 37: better than T4, but not good enough to call a genotype on. Single-nucleotide work needs a genuinely high-fidelity thermostable ligase run hot -- HiFi Taq (21) or, best, Tth (7).

Design rule: put the interrogated base at the padlock's 3' end

Every ligase tested discriminated mismatches far better on the upstream, 3'-OH side of the junction than on the downstream 5'-phosphate side -- Tth tolerated zero upstream mismatches but 7 downstream; T4, 30 versus 48. So design allele-specific padlocks with the discriminating base as the 3'-terminal nucleotide, and give that 3' arm a low Tm (~11°C in the source design) while the 5' arm anchors the probe (~65°C). Getting this backwards costs roughly an order of magnitude in specificity, in any ligase.

Where SplintR sits

SplintR (PBCV-1) is not in this benchmark -- it is the RNA-templated ligase used on the direct-RNA route, a different substrate class. It is well known to tolerate mismatches at the junction, which is precisely why direct RNA has no SNP specificity and why SNP work has to go through cDNA or iLock.

Read the source with its bias in mind

This benchmark is a vendor application note, and Tth -- the winner -- is that vendor's product. The internal ranking that matters most here is nonetheless self-consistent and matches general ligase literature: thermophilic > mesophilic, and 3'-side > 5'-side discrimination. Treat the exact counts as indicative, not absolute.

Picking one

There is no single mandated enzyme -- any of the high-fidelity thermostable ligases will do, and the choice is a trade between the fidelity numbers above and what is already established in the lab:

• Tth DNA Ligase -- the best fidelity in the benchmark (7 single-mismatch mis-ligations, zero on the 3' side) and the most widely used option historically, both in the padlock/SNP literature and in the lab. Works 45--65°C; fidelity improves at the higher end.
• HiFi Taq DNA Ligase (NEB M0647) -- 21 mis-ligations, comfortably in the usable range; convenient ready-to-use format with NAD⁺ already in its buffer. This is what the current SNP-genotyping record runs at 45°C.

Both are NAD⁺-dependent and thermostable, so switching between them changes only the enzyme and its buffer -- the rest of the workflow is unaffected. Start from whichever is on hand; move up the table if a locus proves hard to call.

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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 + high-fidelity<br/>thermostable ligase<br/>(not T4)"]

    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.

If you land on the cDNA branch because you need allele resolution, remember that the strategy alone does not deliver it -- swap the ligase as well. See Ligase Fidelity.

References

1. Thermo Fisher Scientific. Detection of single-nucleotide polymorphisms using a ligation--rolling circle amplification approach. Application note APN-9503200 (2025).
2. New England Biolabs. HiFi Taq DNA Ligase (M0647) Protocol. neb.com/en-us/protocols/hifi-taq-dna-ligase-m0647-protocol
3. 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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1. Thermo Fisher Scientific. Detection of single-nucleotide polymorphisms using a ligation--rolling circle amplification approach. Application note APN-9503200 (2025). Fidelity assessed by capillary electrophoresis of FAM-labelled ligation products across 64 multiplexed substrate pools covering all 256 junction combinations; 50 RPLU per ligase, 20 μL, 30 min, 55°C (thermophilic) or 37°C (mesophilic). ↩

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