Functional DNA origami nanoparticles (DNA-NPs) are used as nanocarriers in a variety of biomedical applications including targeted drug delivery and vaccine development. DNA-NPs can be designed into a broad range of nanoarchitectures in one, two, and three dimensions with high structural fidelity. Moreover, the addressability of the DNA-NPs enables the precise organization of functional moieties, which improves targeting, actuation, and stability. DNA-NPs are usually functionalized via chemically modified staple strands, which can be further conjugated with additional polymers and proteins for the intended application. Although this method of functionalization is extremely efficient to control the stoichiometry and organization of functional moieties, fewer than half of the permissible sites are accessible through staple modifications. In addition, DNA-NP functionalization rapidly becomes expensive when a high number of functionalizations such as fluorophores for tracking and chemical modifications for stability that do not require spatially precise organization are used. To facilitate the synthesis of functional DNA-NPs, we propose a simple and robust strategy based on an asymmetric polymerase chain reaction (aPCR) protocol that allows direct synthesis of custom-length scaffolds that can be randomly modified and/or precisely modified via sequence design. We demonstrated the potential of our strategy by producing and characterizing heavily modified scaffold strands with amine groups for dye functionalization, phosphorothioate bonds for stability, and biotin for surface immobilization. We further validated our sequence design approach for precise conjugation of biomolecules by synthetizing scaffolds including binding loops and aptamer sequences that can be used for direct hybridization of nucleic acid tagged biomolecules or binding of protein targets.

中文翻译：

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用于直接组装功能化 DNA 折纸的定制支架

功能性 DNA 折纸纳米粒子 (DNA-NPs) 在各种生物医学应用中用作纳米载体，包括靶向药物递送和疫苗开发。DNA-NPs 可以设计成具有高结构保真度的一维、二维和三维纳米结构。此外，DNA-NPs 的可寻址性使得功能部分的精确组织成为可能，从而改善了靶向、驱动和稳定性。DNA-NPs 通常通过化学修饰的短链进行功能化，可以进一步与其他聚合物和蛋白质结合以用于预期应用。尽管这种功能化方法对于控制功能部分的化学计量和组织非常有效，但只有不到一半的允许位点可以通过订书钉修饰获得。此外，当使用不需要空间精确组织的大量功能化（例如用于跟踪的荧光团和用于稳定性的化学修饰）时，DNA-NP 功能化会迅速变得昂贵。为了促进功能性 DNA-NP 的合成，我们提出了一种基于不对称聚合酶链反应 (aPCR) 方案的简单而稳健的策略，该方案允许直接合成定制长度的支架，这些支架可以通过序列设计随机修改和/或精确修改. 我们通过生产和表征具有用于染料功能化的胺基、用于稳定性的硫代磷酸酯键和用于表面固定的生物素的高度修饰的支架链来展示我们策略的潜力。