Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification.

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

Spacer-Programmed Two-Dimensional DNA Origami Assembly.

Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of hybridization-mediated 2D DNA origami assembly, DNA spacers play a pivotal role as essential connectors between sticky-end regions and DNA origami units. Here, we demonstrated that programming the spacer length, which determines the binding radius of DNA origami units, could effectively tune sticky-end hybridization reactions to produce distinct 2D DNA origami arrays. Using DNA-PAINT super-resolution imaging, we unveiled the significant impact of spacer length on the hybridization efficiency of sticky ends for assembling square DNA origami (SDO) units. We also found that the assembly efficiency and pattern diversity of 2D DNA origami assemblies were critically dependent on the spacer length. Remarkably, we realized a near-unity yield of ∼98% for the assembly of SDO trimers and tetramers via this spacer-programmed strategy. At last, we revealed that spacer lengths and thermodynamic fluctuations of SDO are positively correlated, using molecular dynamics simulations. Our study thus paves the way for the precision assembly of DNA nanostructures toward higher complexity.

DNA-PAINT Super-Resolution Imaging for Characterization of Nucleic Acid Nanostructures.

Numerous nucleic acid nanostructures of unique addressability and programmability have been fabricated for emerging applications. Structural characterization with atomic force microscopy and electron microscopy can provide information on the structural morphology and precision of these nanostructures. However, either structural information of native nucleic acid nanostructures in hydrated environment or the availability of addressable sites on these nanostructures could not be determined. Alternatively, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) enables direct optical visualization of nucleic acid nanostructures in native forms, as well as evaluation of the accessibility of addressable sites on them. In this Review, the working principle of DNA-PAINT is introduced, followed by the summary on advances of DNA-PAINT characterization of various nucleic acid nanostructures. Finally, the current challenges and prospects for DNA-PAINT characterization are presented. We envision DNA-PAINT to be a potent characterization tool for functional nanomaterials.

DNA Framework-Engineered Long-Range Electrostatic Interactions for DNA Hybridization Reactions.

Long-range electrostatic interactions beyond biomolecular interaction interfaces have not been extensively studied due to the limitation in engineering electric double layers in physiological fluids. Here we find that long-range electrostatic interactions play an essential role in kinetic modulation of DNA hybridizations. Protein and gold nanoparticles with different charges are encapsulated in tetrahedral frameworks to exert diverse electrostatic effects on site-specifically tethered single DNA strands. Using this strategy, we have successfully modulated the hybridization kinetics in both bulk solution and single molecule level. Experimental and theoretical studies reveal that long-range Coulomb interactions are the key factor for hybridization rates. This work validates the important role of long-range electrostatic forces in nucleic acid-biomacromolecule complexes, which may encourage new strategies of gene regulation, antisense therapy, and nucleic acid detection.