TDP-43 is an RNA- and DNA-binding protein initially discovered for binding to the trans-active response element of DNA. It plays a key role in regulating RNA metabolism, mRNA transport, stress granule formation and other cellular processes. Under normal conditions, TDP-43 is found in a soluble form. However, in certain neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), it accumulates in the cytoplasm in the form of insoluble inclusion bodies. TDP-43 belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and is composed of an N-terminal domain (NTD), two RNA recognition motifs (RRM1 and RRM2), and a disordered C-terminal domain (CTD). It also contains a nuclear localization signal (NLS), a nuclear export signal (NES) and several mitochondrial localization signals. In the binding of TDP-43 to nucleic acids, the RRM1 motif has a crucial role by recognizing UG or TG repeats in the sequence, while RRM2 enhances binding specificity. The RRM1 motif contains two essential phenylalanine residues that participate in π-π stacking interactions. There are also eight phenylalanines in the CTD, which may help stabilize the interaction with RNA. We successfully expressed TDP-43 and its mutants by auto-induction in a bacterial T7 expression system, in which some phenylalanine residues in C-terminal domain and in the RRM1 and RRM2 motifs of TDP-43 were mutated to leucine. The proteins were purified using Ni$^{2+}$-affinity chromatography. We also performed in vitro transcription of small RNAs, which were used to analyze protein-RNA binding using the method of microscale thermophoresis (MST).
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