UC Berkeley

The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes.

In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO₄. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved.

Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent cell-targeting delivery vehicles. These agents were generated by selectively functionalizing the interior and exterior surfaces of MS2 with functional molecules and DNA aptamers, respectively, using orthogonal bioconjugation reactions. Interior surface modification was achieved through the incorporation of a uniquely-reactive cysteine residue, while exterior modification occurred via the introduction of the non-natural amino acid p-aminophenylalanine. MS2 capsids possessing interior fluorophores and exterior DNA aptamers targeted to a Jurkat T cell surface receptor were synthesized using this strategy. In cell-binding experiments, these dual-surface modified capsids were shown to bind target cells in an aptamer-dependent manner. In addition, colocalization experiments using confocal microscopy elucidated their cellular internalization pathway.

Following validation of the cell-targeting capabilities of aptamer-MS2 conjugates, a multivalent photodynamic agent was developed for targeted photodynamic therapy. This agent was synthesized by installing singlet oxygen-generating porphyrins on the interior of MS2 capsids possessing DNA aptamers on the exterior. Upon illumination with 415 nm light, these dual-modified capsids were shown to generate cytotoxic singlet oxygen. In cell experiments, these agents were shown to selectively kill Jurkat cells in a heterogeneous cell mixture.