Identifying Factors that Determine Effectiveness of Delivery Agents in Biolistic Delivery Using a Library of Amine-Containing Molecules.

Abstract

Biolistic transfection is a popular and versatile tool for plant transformation. A key step in the biolistic process is the binding of DNA to the heavy microprojectile using a delivery agent, usually a positively charged molecule containing amine groups. Currently, the choice of the commercial delivery agent is mostly limited to spermidine. In addition, the detailed delivery mechanism has not been reported. To help broaden the selection of the delivery agent and reveal the fundamental mechanisms that lead to high delivery performance, a library of amine-containing molecules was investigated. A double-barrel biolistic delivery device was utilized for testing hundreds of samples with much-improved consistency. The performance was evaluated on onion epidermis. The binding and release of DNA were measured via direct high-performance liquid chromatography analysis. This study shows that the overwhelming majority of the amine library performed at the same level as spermidine. To further interpret these results, correlations were performed with thousands of molecular descriptors generated by chemical modeling. It was discovered that the overall charge is most likely the key factor to a successful binding and delivery. Furthermore, even after increasing the amount of the DNA concentration 50-fold to stress the binding capacity of the molecules, the amines in the library continued to deliver at a near identical level while binding all the DNA. The increased DNA was also demonstrated with a Cas9 editing test that required a large amount of DNA to be delivered, and the result was consistent with the previously determined amine performance. This study greatly expands the delivery agent selection for biolistic delivery, allowing alternatives to a commercial reagent that are more shelf-stable and cheaper. The library also offers an approach to investigate more challenging delivery of protein and CRISPR-Cas via the biolistic process in the future.