Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures^1,2,3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates⁴ conditional fluorophores derived from GFP. To extend previous structural studies^5,6 of fluorogenic RNAs, GFP and other fluorescent proteins⁷ to DNA, we characterize Lettuce–fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen⁸ in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding—including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA—and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.

大量研究表明 RNA 分子如何采用复杂的三维 (3D) 结构^1,2,3 。相比之下，DNA 是否可以自组装成复杂的 3D 折叠，能够进行复杂的生物化学，独立于蛋白质或 RNA 伴侣，仍然是个谜。生菜是一种体外进化的 DNA 分子，可结合并激活^{4 个}源自 GFP 的条件荧光团。为了将之前的荧光 RNA、GFP 和其他荧光蛋白⁷的结构研究^5,6扩展到 DNA，我们通过 X 射线晶体学和低温电子显微镜表征了生菜-荧光团复合物。结果表明，53 个核苷酸的 DNA 采用四路连接 (4WJ) 折叠。与 4WJ RNA 中常见的典型 L 形或 H 形结构^不同，生菜的四个茎形成两个同轴堆叠，共线堆积形成荧光团结合的中心 G 四链体。这种折叠通过堆叠、广泛的核碱基氢键（包括通过桥接 DNA 主要同轴堆叠的连续层的不寻常的对角堆叠碱基）以及单价和二价阳离子的协调来稳定。总体而言，该结构比许多同等大小的 RNA 更紧凑。 Lettuce 展示了 DNA 如何在不使用类似 RNA 三级相互作用的情况下形成复杂的 3D 结构，并表明对复杂 DNA 的分析将带来核酸组织的新原理。