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

Abstract

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.

Keywords: ODN conjugate purification; bioconjugation; click chemistry; fluorescence reporters; nucleic acid nanoparticles.

Figure 1

Coupling of ODN alkyne with coumarin azide utilizing “click” chemistry and product analysis. (A) Generalized reaction diagram interaction between 3-azido-7-hydroxycoumarin and 5′-Hexynyl-DNA in a 2:1 ratio in the presence of Cu(I) catalyst. (B) Irradiation of the DNA-Cou conjugate product and control samples by UV lamp 365 nm. (C) Denaturing 32% PAGE demonstrating band shifts between DNA alkyne (reactant) and DNA-Cou (product).

Figure 2

Coupling of 5′-azido-DNA (ODN-azide) with Cy3-alkyne by “click” chemistry and product formation investigation. (A) Generalized reaction diagram interaction between Cy3-alkyne and 5′-azido-DNA in 2:1 ratio in the presence of Cu(I) catalyst. (B) Denaturing 32% PAGE demonstrating band shifts between reactants (Cy3 and DNA azide) and product (DNA-Cy3).

Figure 3

Illustration of membrane filtration principle based on MWCO centrifugal device and fraction analysis. (A) Schematic diagram of the centrifugation steps to evacuate unconjugated reactants and buffer exchange of the post-reaction mixture. Fluorescence assays of the collected fraction of the DNA-coumarin (B) and DNA-Cy3 (C) coupling reactions.

Figure 4

Hybridization of ODN conjugates with triangular-shaped nanoparticles. (A) Secondary structure and sequences of the DNA nanoconstruct representing three sticky-end regions complementary to the sequence of DNA-dyes. (B) Temperature-induced UV-absorbance profile of 1 µM DNA 12-base pairs in TMS buffer. Error corresponds to standard deviation in the mean. (C) Native PAGE analysis of the hybridization products upon saturation of the triangle nanoparticles with increasing number of DNA-conjugates to satisfy 1:0, 1:1, 1:2, and 1:3 stoichiometry ratios. (D). Fluorescence spectra of the triangular nanoparticles containing different numbers of the DNA-coupled dyes.