Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
Bioconjug Chem. 2021 Jan 20;32(1):94-98. doi: 10.1021/acs.bioconjchem.0c00644. Epub 2020 Dec 13.
DNA origami nanostructures generally require a single scaffold strand of specific length, combined with many small staple strands. Ideally, the length of the scaffold strand should be dictated by the size of the designed nanostructure. However, synthesizing arbitrary-length single-stranded DNA in sufficient quantities is difficult. Here, we describe a straightforward and accessible method to produce defined-length ssDNA scaffolds using PCR and subsequent selective enzymatic digestion with T7 exonuclease. This approach produced ssDNA with higher yields than other methods and without the need for purification, which significantly decreased the time from PCR to obtaining pure DNA origami. Furthermore, this enabled us to perform true one-pot synthesis of defined-size DNA origami nanostructures. Additionally, we show that multiple smaller ssDNA scaffolds can efficiently substitute longer scaffolds in the formation of DNA origami.
DNA 折纸纳米结构通常需要一条特定长度的单一支架链，以及许多短的订书钉链。理想情况下，支架链的长度应该由设计的纳米结构的大小决定。然而，大量合成任意长度的单链 DNA 是很困难的。在这里，我们描述了一种使用 PCR 并随后用 T7 外切酶进行选择性酶切来制备特定长度 ssDNA 支架的简单易行的方法。与其他方法相比，该方法产生的 ssDNA 产量更高，且无需纯化，这大大缩短了从 PCR 到获得纯 DNA 折纸的时间。此外，这使我们能够真正实现了具有确定尺寸的 DNA 折纸纳米结构的一锅合成。此外，我们还表明，在 DNA 折纸的形成中，多个较小的 ssDNA 支架可以有效地替代较长的支架。
