Department of Chemistry, Ball State University, Muncie, IN 47306, USA.
Int J Mol Sci. 2023 Mar 2;24(5):4797. doi: 10.3390/ijms24054797.
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.
核酸治疗包括将小分子药物与核酸寡聚物缀合，以克服溶解性和这些药物分子进入细胞的效率低下的挑战。“点击”化学因其简单性和高缀合效率而成为流行的缀合方法。然而，寡核苷酸缀合的主要缺点是产物的纯化，因为传统上使用的色谱技术通常耗时费力，需要大量的材料。在此，我们介绍了一种简单快速的纯化方法，使用分子量截止（MWCO）离心方法分离过量的未缀合小分子和有毒催化剂。作为概念验证，我们采用“点击”化学将 Cy3-炔基部分缀合到叠氮基功能化的寡脱氧核苷酸（ODN）上，以及将香豆素叠氮化物缀合到炔基功能化的 ODN 上。对于 ODN-Cy3 和 ODN-香豆素，计算得到的缀合产物的产率分别为 90.3±0.4%和 86.0±1.3%。通过荧光光谱和凝胶迁移分析对纯化产物的分析表明，报告分子在 DNA 纳米颗粒内的荧光强度大幅度增加了多倍。这项工作旨在展示一种小规模、经济高效且强大的方法，用于纯化核酸纳米技术应用中的 ODN 缀合物。
