Pattanayak Sankha, Vázquez-Maldonado Luis Angel, Deiters Alexander, Chen James K
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, United States.
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, United States.
Methods Enzymol. 2019;624:69-88. doi: 10.1016/bs.mie.2019.04.007. Epub 2019 Apr 25.
Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms. While cMOs are usually triggered by light of a single wavelength, the introduction of spectrally distinct chromophores can enable combinatorial regulation of multiple genes. This chapter describes the general principles and methods of wavelength-selective cMO design and synthesis from commercially available reagents. Synthetic protocols for the linkers and the two-step cMO assembly are described in detail, as well as the microinjection and photoactivation techniques. Following these protocols, spectrally separated cyclic cMOs for multiple genes of interest can be prepared, enabling their inhibition in zebrafish embryos and other animal models.
笼锁吗啉代寡核苷酸（cMOs）是发育生物学中有用的研究工具，因为它们能够在整个生物体中对基因表达进行时空控制。虽然cMOs通常由单一波长的光触发，但引入光谱不同的发色团可以实现对多个基因的组合调控。本章介绍了从市售试剂设计和合成波长选择性cMO的一般原理和方法。详细描述了连接子的合成方案和两步cMO组装，以及显微注射和光激活技术。按照这些方案，可以制备用于多个目标基因的光谱分离的环状cMO，从而能够在斑马鱼胚胎和其他动物模型中抑制这些基因。
