Department of Cell Biology and Neurosciences, Rutgers University, Piscataway, NJ, USA.
Institute of Molecular and Cellular Biology, Goethe University, Frankfurt am Main, Germany.
Methods Mol Biol. 2022;2533:181-197. doi: 10.1007/978-1-0716-2501-9_11.
Cellular RNAs, both coding and noncoding, contain several chemical modifications. Both ribose sugars and nitrogenous bases are targeted for these chemical additions. These modifications are believed to expand the topological potential of RNA molecules by bringing chemical diversity to otherwise limited repertoire. Here, using ribosomal RNA of yeast as an example, a detailed protocol for systematically mapping various chemical modifications to a single nucleotide resolution by a combination of Mung bean nuclease protection assay and RP-HPLC is provided. Molar levels are also calculated for each modification using their UV (254 nm) molar response factors that can be used for determining the amount of modifications at different residues in other RNA molecules. The chemical nature, their precise location and quantification of modifications will facilitate understanding the precise role of these chemical modifications in cellular physiology.
细胞 RNA，无论是编码的还是非编码的，都含有几种化学修饰。核糖和含氮碱基都可以作为这些化学修饰的靶点。这些修饰被认为通过为原本有限的库带来化学多样性来扩展 RNA 分子的拓扑潜力。在这里，我们以酵母核糖体 RNA 为例，提供了一种详细的方案，通过绿豆核酸酶保护分析和反相高效液相色谱（RP-HPLC）的组合，以单核苷酸分辨率系统地对各种化学修饰进行定位。还使用它们的 UV（254nm）摩尔响应因子计算了每种修饰的摩尔水平，这些因子可用于确定其他 RNA 分子中不同残基的修饰量。这些化学修饰的化学性质、精确位置和定量分析将有助于理解这些化学修饰在细胞生理学中的精确作用。
