Shao Yaming, Huang Hao, Qin Daoming, Li Nan-Sheng, Koide Akiko, Staley Jonathan P, Koide Shohei, Kossiakoff Anthony A, Piccirilli Joseph A
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA.
J Mol Biol. 2016 Oct 9;428(20):4100-4114. doi: 10.1016/j.jmb.2016.08.029. Epub 2016 Sep 2.
Antibodies that bind RNA represent an unrealized source of reagents for synthetic biology and for characterizing cellular transcriptomes. However, facile access to RNA-binding antibodies requires the engineering of effective Fab libraries guided by the knowledge of the principles that govern RNA recognition. Here, we describe a Fab identified from a minimalist synthetic library during phage display against a branched RNA target. The Fab (BRG) binds with 20nM dissociation constant to a single-stranded RNA (ssRNA) sequence adjacent to the branch site and can block the action of debranchase enzyme. We report the crystal structure in complex with RNA target at 2.38Å. The Fab traps the RNA in a hairpin conformation that contains a 2-bp duplex capped by a tetraloop. The paratope surface consists of residues located in four complementarity-determining regions including a major contribution from H3, which adopts a helical structure that projects into a deep, wide groove formed by the RNA. The amino acid composition of the paratope reflects the library diversity, consisting mostly of tyrosine and serine residues and a small but significant contribution from a single arginine residue. This structure, involving the recognition of ssRNA via a stem-loop conformation, together with our two previous structures involving the recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of minimalist libraries biased with tyrosine, serine, glycine, and arginine to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy.
能结合RNA的抗体是合成生物学和细胞转录组表征中尚未开发的试剂来源。然而，要轻松获得RNA结合抗体，需要在掌握RNA识别原理的指导下构建有效的Fab文库。在此，我们描述了一种在噬菌体展示过程中从针对分支RNA靶标的简约合成文库中鉴定出的Fab。该Fab（BRG）以20nM的解离常数与分支位点相邻的单链RNA（ssRNA）序列结合，并能阻断去分支酶的作用。我们报道了其与RNA靶标复合物在2.38Å分辨率下的晶体结构。该Fab将RNA捕获在发夹构象中，该构象包含一个由四环封闭的2碱基对双链体。互补决定区表面由位于四个互补决定区的残基组成，其中H3的贡献较大，H3采用螺旋结构，伸入由RNA形成的深而宽的凹槽中。互补决定区的氨基酸组成反映了文库的多样性，主要由酪氨酸和丝氨酸残基组成，单个精氨酸残基也有少量但显著的贡献。这种通过茎环构象识别ssRNA的结构，与我们之前涉及识别RNA发夹环和RNA三级结构的两个结构一起，揭示了用酪氨酸、丝氨酸、甘氨酸和精氨酸偏向的简约文库形成针对特定RNA构象和不同RNA结构层次水平的结合表面的能力。
