Bioconjugation of Functionalized Oligodeoxynucleotides with Fluorescence Reporters for Nanoparticle Assembly.

In the field of nucleic acid nanotechnology and therapeutics, there is an imperative need to improve the oligodeoxynucleotides' (ODNs) properties by either chemical modification of the oligonucleotides' structure or to covalently link them to a reporter or therapeutic moieties that possess biologically relevant properties. The chemical conjugation can thus significantly improve the intrinsic properties not only of ODNs but also reporter/therapeutic molecules. Bioconjugation of nucleic acids to small molecules also serves as a nano-delivery facility to transport various functionalities to specific targets. Herein, we describe a generalized methodology that deploys azide-alkyne cycloaddition, a click reaction to conjugate a cyanine-3 alkyne moiety to an azide-functionalized ODN 12-mer, as well as 3-azido 7-hydroxycoumarin to an alkyne functionalized ODN 12-mer.

Effective, Rapid, and Small-Scale Bioconjugation and Purification of "Clicked" Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization.

Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. "Click" chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed "click" chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications.