Effect of Local Heterogeneities on Single-Layer DNA-Directed Protein Lattices Through Non-Averaged Single-Molecule 3D Structure Determination

TL;DR

This study uses advanced imaging to understand how structural flexibility affects DNA-based 2D protein lattices and suggests ways to improve their design.

Contribution

The study introduces non-averaged 3D structure determination of DNA-origami lattices using IPET, revealing the molecular origin of structural heterogeneity.

Findings

Ferritin loading and placement had minimal impact on lattice parameters or order.

DNA cage flexibility and interparticle linkages caused lattice imperfections.

Molecular dynamics simulations suggested ways to improve lattice design.

Abstract

Programmable and self-assembled two-dimensional (2D) protein lattices hold significant potential in synthetic biology, nanoscale catalysis, and biological devices. However, achieving high-order 2D lattices from three-dimensional (3D) nanoscale objects remains challenging due to structural heterogeneity caused by the flexibility and distortions of building blocks and their connectivity in a unit cell, leading to the formation of lattices with imperfections. This flexibility largely limits the analysis of key structural parameters at unit-cell resolutions due to the need to average 3D reconstructions in current methods. Here, we utilized advances in individual-particle cryo-electron tomography (IPET) to analyze the 3D structure of a designed 2D lattice formed by DNA-origami octahedral cages (unit-cell particles) encapsulating ferritin by determining the non-averaged 3D structure of each unit-cell particle. These protein-carrying DNA cages were analyzed at ferritin loading percentages of 100%, 70%, and 0%. Correlation analysis revealed that neither the ferritin loading percentage nor off-centralized placement in cages significantly affected lattice parameters, flexibility, or long-range order. Instead, the soft nature of DNA cages and interparticle linkages were the primary reasons for lattice imperfections. Structural improvements for enhancing lattice orders were evaluated through a series of molecular dynamics simulations. The developed cryo-EM 3D imaging reveals the molecular origin of heterogeneity of DNA-origami 2D lattices and highlights a path toward improved lattice designs.