Revealing the structures of megadalton-scale DNA complexes with nucleotide resolution

Thomas Gerling,Pierre Stömmer,Elena M Willner,Massimo Kube,Fabian Kohler,Baki Nagel-Yüksel,Jonas J Funke,Hendrik Dietz,Thomas G Martin,Elija Feigl,Sjors H W Scheres,Maximilian N Honemann

doi:10.1038/s41467-020-20020-7

Thomas Gerling, Pierre Stömmer + Show 10 more

Open Access

https://doi.org/10.1038/s41467-020-20020-7

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Abstract

The methods of DNA nanotechnology enable the rational design of custom shapes that self-assemble in solution from sets of DNA molecules. DNA origami, in which a long template DNA single strand is folded by many short DNA oligonucleotides, can be employed to make objects comprising hundreds of unique DNA strands and thousands of base pairs, thus in principle providing many degrees of freedom for modelling complex objects of defined 3D shapes and sizes. Here, we address the problem of accurate structural validation of DNA objects in solution with cryo-EM based methodologies. By taking into account structural fluctuations, we can determine structures with improved detail compared to previous work. To interpret the experimental cryo-EM maps, we present molecular-dynamics-based methods for building pseudo-atomic models in a semi-automated fashion. Among other features, our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks. Obtaining this higher level of detail is a step forward that now allows designers to inspect and refine their designs with base-pair level interventions.

Highlights

The methods of DNA nanotechnology enable the rational design of custom shapes that selfassemble in solution from sets of DNA molecules
The high degree of flexibility and the wrapped-up shape is in accordance with previous findings from simulations, onsupport atomic force microscopy (AFM), negative-stain electron microscopy (EM), and small-angle X-ray scattering (SAXS) data[17,18,19,20], and should be taken into account for in-solution applications
We developed a viewer tool[34] to form a link between the experimentally determined cryo-EM map, the fitted atomic model, and the strand diagram prepared by the designer to build the DNA origami object under study

Summary

Introduction

The methods of DNA nanotechnology enable the rational design of custom shapes that selfassemble in solution from sets of DNA molecules. Our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks Obtaining this higher level of detail is a step forward that allows designers to inspect and refine their designs with base-pair level interventions. We addressed the problem of accurate structural validation with cryoEM-based methodologies for determining the structures of up to megadalton-scale DNA objects in solution, together with molecular-dynamics-based methods for building pseudoatomic models in a semiautomated fashion. Our methods yield structures that afford improved detail compared to our own previous work[13] and to those of others[8,14] (see Supplementary Table S1), and allow discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and nick sites.

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Conclusion