Before you start

• Read Safety & Compliance and the safety data sheet for every reagent below. The SDS, not this page, governs how a reagent is handled, stored and disposed of.
• Use the personal protective equipment and the waste route your laboratory prescribes for these reagent classes.
• Check this protocol against the materials actually in hand: confirm the intended Seq ID, the product revision, the lot-specific certificate and the instructions supplied with it. Where those differ from this page, the documentation supplied with the material governs.

Strategy

The DEL tag carries the synthesis history of its compound, not the compound's structure. The conventional validation route re-synthesizes the hit off-DNA, which changes the chemistry, and a large share of hits do not reproduce once re-synthesized. By ligating an adapter onto the existing tag and measuring the on-DNA compound directly on a molographic chip, you read the affinity of the same chemical entity that was selected — no re-synthesis, no route change.

Ligation mechanism

Three oligos come together: the DEL compound's double-stranded tag with a single-stranded 3′ overhang, the 5′-phosphorylated adapter that carries the chip-surface complementary sequence, and the splint that bridges the junction. T4 DNA Ligase seals the phosphodiester bond at the nick, joining the tag's 3′-OH to the adapter's 5′-phosphate. Heating then releases the splint, and the construct is ready for direct DDI on an Oligo|Oligo chip.

Protocol

The reaction is run in a default final volume of 80 µL at an adapter : splint : tag molar ratio of 1 : 1.5 : 2. The volume is adjustable as long as every reagent is scaled together.

Materials

• T4 DNA Ligase, 30 U µL⁻¹ (e.g. Thermo Fisher EL0013)
• 10× T4 DNA Ligase buffer — supplies the ATP cofactor
• 50 % PEG solution — molecular-crowding accelerator
• Nuclease-free water (NFW)
• 5′-phospho adapter — provided by lino Biotech, complementary to the sensor chip
• Splint oligonucleotide — ordered by the customer
• DNA-tagged compound — the DEL hit, provided by the customer
• Thermal incubator and centrifuge

Step 1 — Combine and denature

Combine the 5′-phospho adapter, the splint, and the DNA-tagged compound at a 1 : 1.5 : 2 molar ratio — for example 1 nmol adapter, 1.5 nmol splint, and 2 nmol tagged compound from 100 µM stocks (10 µL, 15 µL, 20 µL). Spin down and hold at 70 °C for 5 min to melt secondary structure, then leave 30 min at room temperature to cool. Over the cool-down the splint hybridizes across the tag overhang and the adapter 5′ end, seating the nick in register.

Step 2 — Add the ligation mix and ligate

Add 8 µL of 10× T4 DNA Ligase buffer (1× final), 8 µL of 50 % PEG solution, and 1 µL of T4 DNA Ligase (30 U), then top up to 80 µL with nuclease-free water. Spin down and incubate at 37 °C for 2 h — or, alternatively, overnight at 22 °C. The 50 % PEG crowds the reaction and drives the bimolecular join to completion.

Step 3 — Heat-inactivate the ligase and release the splint

Inactivate the ligase at 70 °C for 5 min. The same heat melts the splint off the ligated product. The construct is now compound + dsDNA tag + ssDNA adapter, with the splint free in solution; no purification is required and the mixture can be injected directly.

Step 4 — Inject onto the chip (DDI)

Dilute the ligation product to ≈ 200 nM in running buffer (PBS-T, pH 7.4) and load over the Oligo|Oligo sensor for 5 min at 20 µL min⁻¹. The single-stranded adapter portion hybridizes to its complementary surface oligo on the assigned ridge zone (DDI); free splint passes through without binding. Wash to baseline before starting the analyte injection cycle. To immobilize several compounds at once, combine multiple ligation mixtures — each on its own adapter sequence — and inject them together.

Reference protocol parameters

Chip immobilization (DDI)

The Oligo|Oligo sensor presents a sequence-specific capture oligo on each ridge zone and the backfilling capture oligo (Seq ID 00) on the grooves. The single-stranded adapter portion of the ligated construct hybridizes to its assigned ridge zone in seconds — no chemistry, no covalent step. Up to 64 zones can carry distinct compounds in parallel, one per adapter sequence. For chip-and-adapter selection in the broader workflow, see the interactive decision tree — the DEL / 5′-phosphate branch lands here.

QC and acceptance

The acceptance criterion for the ligation is the appearance of the full-length product; the reaction is expected to convert ≈ 80 % of the adapter.

• Product band. On denaturing PAGE the ligated construct runs above the starting adapter by the length of the tag. The criterion is a clear band at the expected shifted position; residual adapter at the original position is the unreacted fraction, which is expected to be around 10 %.
• Splint and ligase controls. Reactions without the splint and without ligase, run alongside, show that the shift comes from ligation rather than from a mobility artifact of the components.

The DEL tag is deliberately in excess, so unligated tag remains in the mixture by design; it carries no adapter and therefore does not hybridize to the chip, so it neither contributes signal nor competes for the surface. The species that matters on-chip is the ≈ 10 % of free adapter, which is tolerable at the immobilization dilution.

Practical notes

Troubleshooting