An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.