Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3–4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.

长非编码RNA（lncRNA）是最近发现的转录物，可调节重要的细胞过程，例如细胞分化和DNA复制，并且与疾病至关重要。尽管lncRNA的3D结构是其功能的关键决定因素，但迄今为止，lncRNA的空前的分子复杂性使其无法在高分辨率下进行3D结构表征。因此，为这些具有挑战性的目标进行生物化学和生物物理表征的新颖方法至关重要。在这里，我们提出了一个协议，该协议将非变性lncRNA纯化与溶液内流体动力学分析和单颗粒原子力显微镜（AFM）成像相集成，以产生高度均一的lncRNA制剂，并以约15Å的分辨率可视化其3D拓扑结构。我们的协议适用于对具有生物学活性构象的lncRNA进行成像，并适合测量已通过基于细胞的功能测定法鉴定的功能失活突变体的结构缺陷。一旦针对所选的特定靶lncRNA进行了优化，我们的方案将从克隆到AFM成像在3-4周内完成，并且可以由训练有素的熟悉RNA处理并得到AFM支持的研究人员使用最新的生化和生物物理仪器来实施和小角度X射线散射（SAXS）专家。