A recent study in Communications Chemistry shows how a research lab at AMOLF in the Netherlands optimized DNA–protein coupling to study dynamic processes requiring high forces and long-term assessments. Using their C-Trap® Optical Tweezers – Fluorescence Microscopy system, the team showed the superiority of a ligation-based strategy to create long and stable DNA handles.
The study is a further step towards improving protein tethering that requires long and robust DNA handles while simultaneously observing protein dynamics. Congratulations to Prof. Dr. Sander Tans, his lab, and all the authors involved in this work!
Avellaneda et al. first confirmed the higher efficiency of coupling shorter DNA anchors (20 nucleotides) to a protein compared with longer anchors (34 and 40 nucleotides). They were able to generate DNA handles of different lengths that link to the anchors through a DNA template-based exonuclease digestion and partial re-synthesis.
Subsequently, ligating the product to the anchors yielded superior mechanical stability compared with products from existing hybridization protocols. The researchers found that, while hybridized tethers broke at 47 pN, they could pull ligation-derived tethers with forces above 60 pN without breaking them. On top of that, the ligation approach enabled them to use longer DNA handles (e.g., 5 kbp) and prevent parasitic signals from the beads, as observed through correlative confocal microscopy imaging of fluorescent proteins.
These findings represent an essential step towards optimizing protein dynamics studies on large complexes or measuring features that require high forces, long-term assessments, and simultaneous imaging.
For more information, you can read the full article entitled “Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling” in the journal Communications Chemistry.
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