(182d) Automated mRNA Display for Rapid, Affordable, Target-Directed Reagent Development

As more potential cancer targets are being identified, there is a pressing demand for affinity reagents that can specifically bind to them. These reagents are essential to further research on the targets for better cancer diagnosis and treatments. The conventional reagent development methods are unreliable, slow, and expensive to perform. As a result, reagent development has been limited to large research facilities, greatly hindering our advancement in next-generation technologies to fight cancer. In light of these challenges, we set out to build a new reagent development pipeline through automating mRNA display. mRNA display is a powerful technology that screens a library with trillions of members for ultra-high affinity molecules. The target-directed reagents are developed through a cyclic in vitro evolution process that is typically repeated 5-15 times. By automating mRNA display, we aim to transform the technically challenging biochemical process into a broadly-accessible toolset.

To automate mRNA display, we converted the cyclic process into a network of functional microfluidic components using programmable equipment. The microfluidic components are fabricated using a bench-top 3D printer, which enables straightforward, rapid, and low-cost prototyping of the devices throughout the system development process. For instance, we designed a microfluidic enrichment device (MFED) for automating the bead-based purification steps in mRNA display. In bead-based purifications, peptides are allowed to bind the bead-immobilized target and the ones that are not bound to the target are removed. When performed manually, the removal of unbound peptides is done by repeatedly washing the beads using centrifugal filters. This approach, however, is not applicable to automation. Instead, we automated the purification steps using the MFED which immobilizes the target-bead complexes within a microfluidic channel. The device enables automated bead washing in a continuous-flow manner.

To benchmark the performance of the automated system, we performed controlled enrichments using previously prepared ligands against B-cell lymphoma-extra large (Bcl-x_L), a pro-survival cancer protein. We demonstrated that the MFED considerably outperforms the manual technique, with higher enrichment capability per round of selection. Using the MFED, the automated system can further accelerate mRNA display and lower the cost to generate reagents because of its superior performance in selection.

The automated mRNA display has transformative potential in addressing the current challenges of reagent development. By combining microfluidic automation and mRNA display, we aim to create an inexpensive platform for ligand discovery, expand the use of reagents in the biomedical and biopharma community, and thereby accelerate the evolution of cancer diagnostic and therapeutic tools.