Delivery of DNA Encoded Libraries (DELs) Intracellularly via Mechanoporation

Who this is for:

Drug discovery groups and CROs

Opportunities

Direct to Biology DELs: Intracellular delivery to live cells enables target proteins to be in their native cellular context and drive desired phenotypes
More relevant hits: Higher probability that identified hits will continue to bind target in the desired cellular context

Results Obtained

Delivery of fluorescently tagged DEL libraries to iPSCs and HeLa cells
Verification that delivery does not cause bias and delivers the full diversity of compounds into cells

How We Did It

Portal uses mechanoporation to deliver virtually any cargo to diverse cell types while maintaining cell health. This enables intracellular delivery of impermeable molecules without the complications of electroporation or other delivery methods. Learn more about mechanoporation here!

Fig 1: Induced pluripotent stem cells (iPSCs) were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting.

Fig 2: HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting.

Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells

iPSCs and HeLa’s were boosted with a fluorescently-tagged DNA encoded library (DEL) probe. Strong delivery efficiency was shown when measured using flow cytometry.

Quantification of DEL Delivery into HEK293T Cells via qPCR

HEK293T cells were treated with DNA encoded with a DNA-encoded library (DEL) conjugated to a small molecule, and intracellular DEL concentrations were quantified by qPCR. A standard curve generated using the same oligo was used to calculate normalized DEL concentrations (nM). Cells receiving a boost showed a significant increase in intracellular DEL levels (~125,000 DEL molecules per cell) compared to both untreated and no-boost conditions.

Fig. 4: The DEL was delivered proportionally into PBMCs with mechanoporation technology. Shown is the Spearman R correlation analysis of the DEL library alone vs the DEL library after being mechanoporated into cells.

Fig. 5: Control showing the replicate of two samples of the same DEL library

Fig 6: DEL control vs a target biased library to demonstrate what a non proportional delivery would look like

DEL Delivered Proportionally into PBMCs with Mechanoporation

PBMCs were mechanoporated in the presence of a diverse DEL. After washing, the cells were lysed and analyzed via sequencing. The DEL was delivered proportionally into PBMCs with mechanoporation. Shown is the Spearman R correlation analysis of DEL features between experimental conditions.
The correlation of diluted DEL sample with the mechanoporated sample, (Fig. 4, R2=0.93) is close to that with the technical replicate of just the DEL library (Fig. 5, R2=0.98). This is drastically greater than that with the purposefully biased target-selected DEL sample (Fig. 6, R2=0.77). The result suggests that the DEL delivered by mechanoporation is proportional to the initial DEL distribution.