[Paper Review] Hairygami: Analysis of DNA Nanostructures' Conformational Change Driven by Functionalizable Overhangs

Matthew Sample, Hao Liu+3 more|arXiv (Cornell University)|Feb 17, 2023

Advanced biosensing and bioanalysis techniquesBiochemistry, Genetics and Molecular Biology49 references3 citations

TL;DR

This study reveals that functionalizable single-stranded DNA overhangs induce curvature in 2D DNA origami tiles due to entropic effects, as shown through oxDNA coarse-grained molecular dynamics simulations and validated experimentally via TEM. The overhangs alter the conformational ensemble in solution, challenging the assumption that origami structures remain flat, with implications for designing robust DNA nanostructures and controlling their shape via overhang engineering.

ABSTRACT

DNA origami is a widely used method to construct nanostructures by self-assembling designed DNA strands. These structures are often used as "pegboards" for templated assembly of proteins, gold nanoparticles, aptamers, and other molecules, with applications ranging from therapeutics and diagnostics to plasmonics and photonics. Imaging these structures using AFM or TEM does not capture their full conformation ensemble as they only show their shape flattened on a surface. However, certain conformations of the nanostructure can position guest molecules into distances unaccounted for in their intended design, thus leading to spurious interactions between guest molecules that are designed to be separated. Here, we use molecular dynamics simulations to capture conformational ensemble of 2D DNA origami tiles and show that introducing single-stranded overhangs, which are typically used for functionalization of the origami with guest molecules, induces a curvature of the tile structure in the bulk. We show that the shape deformation is of entropic origin, with implications for design of robust DNA origami breadboards as well as potential approach to modulate structure shape by introducing overhangs. We then verify experimentally that the DNA overhangs introduce curvature into the DNA origami tiles in divalent as well as monovalent salt buffer conditions. We further experimentally verify that DNA origami functionalized with attached proteins also experience such induced curvature. We provide the developed simulation code implementing the enhanced sampling to characterize conformational space of DNA origami as open source software.

Motivation & Objective

• To investigate how functionalizable DNA overhangs affect the conformational dynamics of 2D DNA origami tiles in solution.
• To address the limitation of surface-based imaging techniques (e.g., AFM, TEM) that fail to capture solution-phase conformational ensembles.
• To determine whether overhangs induce structural deformation in DNA origami beyond their intended design.
• To provide a computational and experimental framework for characterizing the conformational ensemble of DNA nanostructures in bulk solution.
• To enable design principles for robust DNA origami breadboards by understanding overhang-induced shape changes.

Proposed method

• Employed the oxDNA coarse-grained model (version 2) with GPU-accelerated simulations to model 2D DNA origami tiles with varying overhang lengths.
• Used umbrella sampling with harmonic biasing potentials to compute free energy profiles across end-to-end distance (R_ee) as the order parameter.
• Applied the Weighted Histogram Analysis Method (WHAM) to unbias simulation data and reconstruct free energy landscapes with 200 bins.
• Performed Monte Carlo bootstrapping for error estimation on free energy profiles and weighted averages.
• Used polynomial fitting to relate R_ee to the angle of curvature for structural interpretation.
• Validated predictions experimentally using gel electrophoresis and negative staining transmission electron microscopy (TEM).

Figure 1: The addition of overhangs causes a $2$ D DNA origami tile $(a)$ to adopt a curved shape $(b)$ . The origin of the curvature is mainly due to the entropic penalty of the overhang sequences on a flat surface $(c)$ , compared to a curved surface $(d)$ (shown schematically as a side view of an

Experimental results

Research questions

• RQ1 Do functionalizable DNA overhangs induce structural curvature in 2D DNA origami tiles when in solution?
• RQ2 What is the thermodynamic origin of the curvature induced by overhangs—entropic or enthalpic?
• RQ3 How do overhang length and position affect the conformational ensemble of DNA origami in solution?
• RQ4 Can molecular dynamics simulations accurately predict the solution-phase shape of DNA origami with overhangs?
• RQ5 To what extent do surface-based imaging techniques like AFM and TEM fail to represent the true solution conformation of DNA origami?

Key findings

• Single-stranded overhangs induce measurable curvature in 2D DNA origami tiles in solution, with the degree of curvature increasing with overhang length.
• The curvature is driven by entropic effects, as confirmed by free energy profiles showing a preference for curved conformations.
• The free energy landscape reveals a stable curved state with a minimum at R_ee ≈ 30 nm, indicating a preferred bent conformation.
• Simulations with 40 overhangs showed a 2.6-fold increase in sampling efficiency when run in parallel on a single GPU, enabling large-scale conformational analysis.
• Experimental TEM imaging confirmed the presence of curvature in overhang-functionalized origami tiles, validating the simulation predictions.
• The study demonstrates that overhangs can be used as a design element to actively modulate the shape of DNA nanostructures in solution.

Figure 2: $(a)$ Mean structure of twist-corrected rectangular origami with $169$ overhang extensions comprised of twenty nucleotide bases. The arrows indicate the measured end-to-end distance order parameter ( $R_{\rm ee}$ ) used to model the curvature of the structures. Low $R_{\rm ee}$ values corr