Optimizing DNA origami assembly through selection of scaffold sequences that minimise off-target interactions
Abstract
DNA origami is a mainstay of DNA nanotechnology and several efforts have been devoted to understanding how various factors of the self-assembly reaction affect the final yield of the target origami structure. This study analyses how base sequence affects origami yield through the generation of off-target side reactions during selfassembly. Off-target bindings are an under-explored phenomenon and can potentially introduce unwanted assembly barriers and kinetic traps in the origami folding pathway. We developed a multi-objective computational approach that takes a given origami design and scores different scaffold sequences (and their complementary staples) for the prevalence of four different types of off-target binding events. Using our method on DNA origami, we can select ‘bad’ regions of biological sequences (like lambda DNA phage) that, when used as origami scaffold sequences, have an excessive number of off-target side reactions for each shape. We show, using high-resolution atomic force microscopy (AFM), that these scaffold sequences largely fail to fold into the target triangle or rectangle structure in vitro , despite the scaffold sequence having a fully complementary staple set present. Conversely, using our method we can also select ‘good’ regions of biological sequences. These sequences are deficient in off-target reactions and when used as origami scaffolds, fold more successfully into their target structures as characterised by AFM. These results have been validated in “blind” folding experiments at two different laboratories in which the experimenters did not know which scaffolds were good or bad folders. To further investigate assembly behaviour, optical tweezers experiments revealed distinct mechanical response profiles, correlating with scaffold-specific off-target interactions. While variants with higher GC content show a high mean unfolding force, variants with lower off-target binding demonstrated more uniform force-extension curves. Our analysis confirmed that high off-target binding leads to increased structural heterogeneity, as seen in the clustering behaviour of unfolding traces of OT experiments. Over-all, our work demonstrates how the off-target reactions implicit in base sequences can derail the origami self-assembly process if sufficiently prevalent, and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design.
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BibTeX
@article{shirtediss2026optimizing,
title = {{Optimizing DNA origami assembly through selection of scaffold sequences that minimise off-target interactions}},
author = {Ben Shirt-Ediss and Emanuela Torelli and Silvia Adriana Navarro and Hadeel Khamis and Ariel Kaplan and William Trewby and Juan Elezgaray and Nima Moradzadeh-Esmaeili and Michael Haydell and Daniel Keppner and Michael Famulok and Kai Armstrong and Natalio Krasnogor},
journal = {Nature Communications},
year = {2026},
month = may,
doi = {10.1038/s41467-026-73387-4},
url = {https://hal.science/hal-05361468v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Optimizing DNA origami assembly through selection of scaffold sequences that minimise off-target interactions
AU - Ben Shirt-Ediss
AU - Emanuela Torelli
AU - Silvia Adriana Navarro
AU - Hadeel Khamis
AU - Ariel Kaplan
AU - William Trewby
AU - Juan Elezgaray
AU - Nima Moradzadeh-Esmaeili
AU - Michael Haydell
AU - Daniel Keppner
AU - Michael Famulok
AU - Kai Armstrong
AU - Natalio Krasnogor
PY - 2026
DA - 2026/05/26
JO - Nature Communications
DO - 10.1038/s41467-026-73387-4
UR - https://hal.science/hal-05361468v1
AB - DNA origami is a mainstay of DNA nanotechnology and several efforts have been devoted to understanding how various factors of the self-assembly reaction affect the final yield of the target origami structure. This study analyses how base sequence affects origami yield through the generation of off-target side reactions during selfassembly. Off-target bindings are an under-explored phenomenon and can potentially introduce unwanted assembly barriers and kinetic traps in the origami folding pathway. We developed a multi-objective computational approach that takes a given origami design and scores different scaffold sequences (and their complementary staples) for the prevalence of four different types of off-target binding events. Using our method on DNA origami, we can select ‘bad’ regions of biological sequences (like lambda DNA phage) that, when used as origami scaffold sequences, have an excessive number of off-target side reactions for each shape. We show, using high-resolution atomic force microscopy (AFM), that these scaffold sequences largely fail to fold into the target triangle or rectangle structure in vitro , despite the scaffold sequence having a fully complementary staple set present. Conversely, using our method we can also select ‘good’ regions of biological sequences. These sequences are deficient in off-target reactions and when used as origami scaffolds, fold more successfully into their target structures as characterised by AFM. These results have been validated in “blind” folding experiments at two different laboratories in which the experimenters did not know which scaffolds were good or bad folders. To further investigate assembly behaviour, optical tweezers experiments revealed distinct mechanical response profiles, correlating with scaffold-specific off-target interactions. While variants with higher GC content show a high mean unfolding force, variants with lower off-target binding demonstrated more uniform force-extension curves. Our analysis confirmed that high off-target binding leads to increased structural heterogeneity, as seen in the clustering behaviour of unfolding traces of OT experiments. Over-all, our work demonstrates how the off-target reactions implicit in base sequences can derail the origami self-assembly process if sufficiently prevalent, and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design.
SN - 2041-1723
SN - 2041-1723
ER -