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

Area of Science:

• Synthetic biology
• Nanotechnology
• Structural biology

Background:

• Programmable two-dimensional (2D) protein lattices are promising for synthetic biology, catalysis, and devices.
• Achieving high-order 2D lattices from 3D nanoscale objects is difficult due to structural heterogeneity and imperfections.
• Current methods average 3D reconstructions, limiting unit-cell resolution analysis.

Purpose of the Study:

• To analyze the 3D structure of a designed 2D lattice formed by DNA-origami octahedral cages encapsulating ferritin.
• To determine the non-averaged 3D structure of each unit-cell particle using individual-particle cryo-electron tomography (IPET).
• To identify the molecular origins of heterogeneity in DNA-origami 2D lattices.

Main Methods:

• Individual-particle cryo-electron tomography (IPET) to determine non-averaged 3D structures.
• Analysis of DNA-origami octahedral cages with varying ferritin loading (100%, 70%, 0%).
• Correlation analysis to assess the impact of ferritin loading and placement on lattice parameters.

Main Results:

• Ferritin loading percentage and off-centralized placement did not significantly affect lattice parameters, flexibility, or long-range order.
• The primary causes of lattice imperfections were identified as the soft nature of DNA cages and interparticle linkages.
• Molecular dynamics simulations were used to evaluate strategies for improving lattice order.

Conclusions:

• Cryo-electron tomography imaging revealed the molecular basis of heterogeneity in DNA-origami 2D lattices.
• Lattice imperfections stem from DNA cage flexibility and linkage properties, not payload variations.
• The study provides insights for designing more ordered and robust 2D lattices.