Monday, June 12, 2017

DNA origami supported precision measurements of biomolecular interactions and structure

Hendrik Dietz

Programmable self-assembly with DNA origami allows creating custom-shaped nanoscale objects. Through this capacity, DNA origami enables constructing custom instruments to perform precision measurements of molecular interactions and structure, with enhanced control over positioning, orientating and manipulating the molecules under study. In my presentation I will report about a series of experiments in which we exploited this capacity to dissect the weak stacking forces between individual basepairs (1) and the forces that act between pairs of nucleosomes (2).
In experiment (1), we directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from −0.8 to −3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.
In experiment (2), we performed a direct measurement of inter-nucleosomal interactions by integrating two nucleosomes into a DNA origami-based force spectrometer, which enabled sub-nanometer resolution measurements of nucleosome-nucleosome distance frequencies via single particle electron microscopy imaging. From the data we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-ranged (~6 nm) attractive nucleosome pair potential with a minimum of −1.6 kcal/mol close to direct-contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Due to the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional co-factors. Our results contribute to a more accurate description of chromatin, while our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques.

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