Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

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† These authors have contributed equally to this work. AbstractHuman hepatic flavin--containing monooxygenase 3 is a phase I drug--metabolizing enzyme that is responsible for the oxidation of a variety of drugs and xenobiotics. This work reports on a high--throughput rapid colorimetric assay for the screening of substrates or inhibitors of this enzyme. The method is based on the competition of two substrates for access to the active site of hFMO3 whereby the enzymatic product of the first drug converts nitro--5--thiobenzoate (TNB, yellow) to 5,5'--dithiobis(2--nitrobenzoate) (DTNB, colourless). Upon addition of a competing substrate, the amount of detected DNTB is decreased. The assay is validated testing three known substrates of hFMO3, namely benzydamine, tozasertib and tamoxifen. The latter drugs resulted in 41% --55% inhibition. In addition, two other drugs also classified as doping drugs, selegiline and clomiphene, were selected based on their chemical structure similarity to known substrates of hFMO3. These drugs showed 21% and 60% inhibition in the colorimetric assay and therefore were proven to be hFMO3 substrates. LC--MS was used to confirm their N--oxide products. Further characterisation of these newly identified hFMO3 substrates was performed determining their K m and k cat values that resulted to be 314 µM and 1.4 min --1 for selegiline and, 18 µM and 0.1 min --1 for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen--containing compounds as substrates/inhibitors of hFMO3.

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.