A Small Chemical Tweak Makes DNA Build Two Very Different Nanostructures

By Muhammad OsamaReviewed by Susha Cheriyedath, M.Sc.Sep 20 2026

By giving DNA tiles a second molecular interaction beyond base pairing, researchers uncovered how hydrophobic chain length can redirect the sequence of assembly and open new possibilities for programmable nanoscale materials.

Paper: Expanding the DNA Nanotechnology Toolbox: Hierarchical Assembly and Self-Sorting of DNA Tiles With Precisely Controlled Hydrophobic Behavior. Image Credit: Piyaset / Shutterstock

Controlling the assembly of DNA (Deoxyribonucleic Acid) nanostructures beyond conventional base pairing remains a challenge in structural nanotechnology. A recent study published in the journal Angewandte Chemie International Edition has incorporated hydrophobic domains into double-crossover tile motifs to control their assembly.

By varying the number of hexaethylene phosphate (C12) units in the hydrophobic segments, researchers produced different nanoscale structures, including star-shaped networks and spherical nucleic acids (SNAs). This approach adds hydrophobic interactions to the base-pairing rules that guide DNA assembly, offering a route to higher-order structures while reducing reliance on large numbers of unique DNA sequences.

Expanding Programmability with Amphiphilic Polymers

DNA can form predictable structures through complementary Watson-Crick base pairing, making it a valuable building material for nanoscale structures studied for molecular therapies and diagnostics. Yet structures relying solely on complementary base pairing often require many unique sequences, particularly in complex networks such as DNA origami.

To create more intricate structures with fewer sequences, scientists have explored additional non-covalent interactions. One effective approach uses sequence-defined amphiphilic polymers attached to DNA backbones, introducing additional interactions inspired by the cooperative assembly mechanisms used by proteins. This enables higher-order organization.

Methodology for Constructing Hydrophobic DNA Tiles

Researchers designed double-crossover tiles consisting of two parallel helices connected by two four-way junctions. They employed an antiparallel motif with an even number of half-turns between crossover points, which is particularly stable and planar. Each tile complex contained five distinct oligonucleotides. Using solid-phase synthesis and phosphoramidite chemistry, two strands were modified at their 5′ ends with varying numbers of hexaethylene phosphate (C12) units, forming hydrophobic segments of precisely defined length.

To understand how the modified tiles assembled, the study employed thermal annealing by heating the samples to 95 °C, holding for 5 minutes, then cooling from 85 °C to 25 °C at 1 °C per minute. Isothermal incubation at 37 °C was also evaluated. Atomic force microscopy (AFM) was utilized to examine the resulting nanoscale structures. The design was further applied to three-point star tiles carrying two alkyl chains on one arm.

Nile Red, an environmentally sensitive fluorescent dye, was employed to track the formation of hydrophobic domains during assembly. Changes in fluorescence emission during cooling were analyzed to determine the temperatures at which hydrophobic core formation began.

Ultraviolet absorbance at 260 nm was used to measure melting temperatures and assess structural stability. Cyanine-3-labeled indicator strands were added to pre-assembled structures during incubation, and native agarose gel electrophoresis was used to assess strand incorporation and crosslinking. Separate experiments assessed nuclease resistance in 10% fetal bovine serum, while size-exclusion chromatography tested whether human serum albumin disrupted the assemblies.

Assembly Mechanisms and Self-Sorting Dynamics

The results demonstrated that hydrophobic chain length significantly influenced the order of DNA tile assembly. With shorter chains containing four or six hexaethylene units, the base-pairing temperature was higher than the hydrophobic assembly temperature. Base pairing occurred first, producing individual double-crossover tiles that then assembled into three- or four-arm nanostars. The four-unit variants also formed extended networks, which the authors propose were likely driven by π-π stacking interactions at blunt tile ends.

For longer chains containing twelve or sixteen repeating units, hydrophobic assembly occurred at higher temperatures than base pairing. Hydrophobic association initially produced spherical nucleic acids, bringing the modified strands into proximity on the particle surface. Further cooling then promoted base pairing, resulting in crosslinked spherical nucleic acid structures. Agarose gel electrophoresis indicated that these structures resisted incorporation of externally added Cyanine-3-labeled indicator strands after 12 hours of incubation, suggesting that the structures were highly crosslinked and resistant to strand exchange.

The crosslinked spherical nucleic acids showed high nuclease resistance in a medium containing 10% fetal bovine serum. Size-exclusion chromatography revealed that non-crosslinked SNAs dissociated after exposure to excess human serum albumin, while the crosslinked SNAs remained intact. When mixtures containing the same DNA sequences but different hydrophobic chain lengths were annealed in a single solution, the components separated into distinct nanostar and spherical populations, indicating narcissistic self-sorting, in which components preferentially assembled with counterparts carrying the same hydrophobic chain length rather than forming mixed assemblies. Isothermal incubation at 37 °C, by contrast, produced a single population, supporting the role of thermal annealing in the self-sorting process.

Potential for Future Targeted Drug Delivery

The engineered DNA structures show potential for biomedical applications, including drug delivery. Their resistance to nuclease degradation and albumin-mediated dissociation suggests that crosslinking could help create more stable DNA-based carriers under biologically relevant conditions. The study did not evaluate therapeutic cargo loading, release, cellular delivery, or in vivo performance; these applications require further investigation.

The incorporation of single-stranded toehold domains provides a mechanism for controlling structural changes. In the study, a trigger oligonucleotide bound the engineered toehold and displaced a backbone strand involved in surface crosslinking, shifting the SNA toward a non-crosslinked state. This programmable strand displacement could be developed as a strategy to alter the stability or accessibility of structures in response to specific molecular signals, potentially enabling conditional cargo release in future studies; release itself was not tested here.

Future Directions in DNA Nanostructure Design

Overall, the study demonstrates that adding sequence-defined hydrophobic modifiers to DNA tiles can effectively control their assembly. By adjusting the balance between base pairing and hydrophobic interactions, researchers produced various morphologies, including networked star and crosslinked spherical structures. This approach offers a route to complex structures while reducing reliance on large numbers of unique DNA sequences, thereby enabling the integration of diverse non-covalent interactions in DNA nanostructure design. The authors propose integrating DNA aptamer sequences into the toehold and backbone domains to recognize a broader range of disease markers and improve targeting in future delivery studies.

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