MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA–protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins.* In the article “DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices” Ming Liu, Saeid Movahed, Saroj Dangi, Hai Pan, Parminder Kaur, Stephanie M. Bilinovich, Edgar M. Faison, Gage O. Leighton, Hong Wang, David C. Williams Jr. and Robert Riehn demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), they determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops.* Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernible large-scale impact of protein binding. The authors of the article demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.* For atomic force microscopy (AFM), the authors used a 7,163-bp linear DNA substrate which contains a 1,697-bp methylated CpG-rich region that is flanked by 2,742-bp and 2,724-bp CpG-free regions. For MeCP2, the DNA substrate and the protein were diluted in AFM imaging buffer (HEPES 20 mM, Mg(OAc)210mM, NaCl 100mM, pH 7.5), mixed together and deposited on freshly peeled mica. For MBD2FLsc, the authors describe how they first mixed the protein and DNA and then diluted the sample in AFM buffer before deposition. The final MeCP2 concentration deposited on mica was 7.5nM, and the MBD2FLsc concentration was 14nM. The mica samples were then washed with filtered deionized water and dried with nitrogen.* NANOSENSORS™ PointProbe® Plus PPP-FMR AFM probes ( ≈2.8N/m) were used to image the sample at a scan resolution of 5.9nm and a scan rate of 3μm/s.* https://www.nanosensors.com/pointprobe-plus-force-modulatio… Please have a look at the NANOSENSORS blog for the full citation and a direct link to the full article. #MeCP2 #DNA #走査型走査型プローブ顕微鏡 #SPMプローブ #AtomicForceMicroscopy #lifescience