Galvan Angelica Rose, Green Christopher M, Hooe Shelby L, Oktay Esra, Thakur Meghna, Díaz Sebastián A, Veneziano Remi, Medintz Igor L, Mathur Divita
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States; Fischell Department of Bioengineering, College of Engineering, University of Maryland, College Park, Maryland 20742, United States.
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States.
ACS Appl Nano Mater. 2024 Jun 14;7(11):12891-12902. doi: 10.1021/acsanm.4c01456. Epub 2024 May 30.
DNA nanotechnology has made initial progress toward developing gene-encoded DNA origami nanoparticles (NPs) that display potential utility for future gene therapy applications. However, due to the challenges involved with gene delivery into cells including transport through the membrane, intracellular targeting, and inherent expression of nucleases along with interference from other active proteins, it can be difficult to more directly study the effect of DNA NP design on subsequent gene expression. In this work, we demonstrate an approach for studying the expression of gene-encoding DNA origami NPs without the use of cells. We utilize a pure -derived cell-free transcription-translation (TXTL) system, which is composed of optimized components from bacterial expression, for benchtop studies to assess how the promoter sequence in conjunction with structural design of the DNA NP template affects gene expression. The gene for an optimized was first amplified into a single-stranded (ss) scaffold strand and then folded into a 12-helix bundle DNA NP with exogenous staple strands as a test platform. Using luciferase-based bioluminescence assays to characterize the relative protein expression level, it was found that the gene can still be transcribed when folded, albeit at a lower rate than the double-stranded DNA gene segment. On comparing three variants of DNA NP with different promoter configurations, results indicate that a promoter designed to remain in ssDNA form has reduced protein expression from the DNA NP, and replacing the promoter sequence with an arbitrary sequence significantly lowers protein expression. This work demonstrates the power inherent in cell-free TXTL systems as an aid to study the gene expression capabilities of DNA NPs toward design and development of future applications.
DNA纳米技术在开发基因编码的DNA折纸纳米颗粒（NPs）方面已取得初步进展，这些纳米颗粒在未来的基因治疗应用中显示出潜在的效用。然而，由于将基因递送至细胞中存在诸多挑战，包括穿过细胞膜的转运、细胞内靶向、核酸酶的固有表达以及来自其他活性蛋白的干扰，因此更直接地研究DNA纳米颗粒设计对后续基因表达的影响可能会很困难。在这项工作中，我们展示了一种无需使用细胞即可研究基因编码DNA折纸纳米颗粒表达的方法。我们利用一种纯衍生的无细胞转录-翻译（TXTL）系统，该系统由来自细菌表达的优化成分组成，用于台式研究，以评估启动子序列与DNA纳米颗粒模板的结构设计如何影响基因表达。首先将一种优化的基因扩增为单链（ss）支架链，然后与外源短链一起折叠成一个12螺旋束DNA纳米颗粒作为测试平台。使用基于荧光素酶的生物发光测定法来表征相对蛋白质表达水平，发现该基因在折叠时仍可转录，尽管转录速率低于双链DNA基因片段。在比较具有不同启动子构型的三种DNA纳米颗粒变体时，结果表明，设计为保持单链DNA形式的启动子会降低DNA纳米颗粒的蛋白质表达，而用任意序列替换启动子序列会显著降低蛋白质表达。这项工作证明了无细胞TXTL系统在辅助研究DNA纳米颗粒的基因表达能力以用于未来应用的设计和开发方面所具有的内在力量。
