DataSheet1_Selection and Modelling of a New Single-Domain Intrabody Against TDP-43.docx

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated to deteriorating motor and cognitive functions, and short survival. The disease is caused by neuronal death which results in progressive muscle wasting and weakness, ultimately leading to lethal respiratory failure. The misbehaviour of a specific protein, TDP-43, which aggregates and becomes toxic in ALS patient’s neurons, is supposed to be one of the causes. TDP-43 is a DNA/RNA-binding protein involved in several functions related to nucleic acid metabolism. Sequestration of TDP-43 aggregates is a possible therapeutic strategy that could alleviate or block pathology. Here, we describe the selection and characterization of a new intracellular antibody (intrabody) against TDP-43 from a llama nanobody library. The structure of the selected intrabody was predicted in silico and the model was used to suggest mutations that enabled to improve its expression yield, facilitating its experimental validation. We showed how coupling experimental methodologies with in silico design may allow us to obtain an antibody able to recognize the RNA binding regions of TDP-43. Our findings illustrate a strategy for the mitigation of TDP-43 proteinopathy in ALS and provide a potential new tool for diagnostics.

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis，简称ALS）是一种与运动和认知功能衰退以及短暂生存期相关的神经退行性疾病。该疾病由神经元死亡引起，导致肌肉逐渐萎缩和无力，最终导致致命的呼吸衰竭。在ALS患者神经元中，特定蛋白质TDP-43的异常行为，即其聚集成毒性，被认为是病因之一。TDP-43是一种参与核酸代谢相关多种功能的DNA/RNA结合蛋白。TDP-43聚集体被隔离是一种可能的疗法，可以缓解或阻断病理过程。在此，我们描述了从骆驼纳米抗体库中选择并表征了一种针对TDP-43的新型细胞内抗体（intrabody）。所选intrabody的结构通过计算机模拟预测，并利用该模型提出了能够提高其表达产量的突变，从而促进其实验验证。我们展示了如何将实验方法与计算机辅助设计相结合，以获得能够识别TDP-43的RNA结合区域的抗体。我们的发现阐明了一种缓解ALS中TDP-43蛋白病的方法，并为诊断提供了潜在的新的工具。