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 gives a 70–90 % false-positive rate. 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 dsDNA tag with a 3′ ssDNA overhang and 5′-phosphate, the splint that bridges the junction, and the adapter that carries the chip-surface complementary sequence. T4 DNA Ligase seals the phosphodiester bond at the nick. Once the splint dissociates, the construct is ready for direct DDI on an Oligo|Oligo chip.

Protocol

Step 1 — Verify the DEL hit oligo

Confirm the DEL tag has a 5′-phosphate and a defined 3′ overhang sequence (the ligation target for the splint). If the supplier delivers the tag without phosphorylation, treat it with T4 polynucleotide kinase (PNK) plus 1 mM ATP at 37 °C for 30 min and heat-inactivate for 20 min at 65 °C before continuing.

Step 2 — Anneal splint to adapter

Combine the splint and the adapter at equimolar ratio (1 µM each) in T4 Ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM ATP, 10 mM DTT). Heat to 70 °C for 5 min and cool to 16 °C at 0.1 °C s⁻¹ in a thermocycler. This pre-anneal seats the splint onto the adapter so the tag has a clean docking site in the next step.

Step 3 — Add the DEL hit and ligate

Mix the pre-annealed splint:adapter duplex with the DEL hit at 1:1 molar ratio (typical working concentration 0.5–1 µM each). Add T4 DNA Ligase to 20 U µL⁻¹ final and incubate at 16 °C for 1 h. The splint hybridizes to the tag overhang on one side and to the adapter on the other, presenting a single nick that the ligase seals (5′-P on the tag joined to 3′-OH of the adapter).

Step 4 — Heat-inactivate the ligase and remove the splint

Heat the reaction to 65 °C for 10 min to inactivate the ligase and to dehybridize the splint from the ligated product. Hold at 65 °C for the entire time — premature cooling lets the splint re-bind. Cool to room temperature on the bench. The construct is now compound + dsDNA tag + ssDNA adapter; the splint is free in solution and is washed out at the chip step.

Step 5 — Inject onto the chip

Dilute the ligation product to 50–200 nM in PBS-T (pH 7.4) and inject over the Oligo|Oligo sensor at the rate specified by your plex protocol. 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.

Optimal parameters at a glance

Chip immobilization (DDI)

The Oligo|Oligo sensor presents a sequence-specific capture oligo on each ridge zone and an inert oligo 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 16 zones (standard DEL screen) or 54 zones (high-throughput screen) can carry distinct compounds in parallel. For chip-and-adapter selection in the broader workflow, see the interactive decision tree — the DEL / 5′-phosphate branch lands here.

Practical notes

Troubleshooting