Toehold-Mediated Selective Assembly of Compact Discrete DNA Nanostructures

Abstract

Production of small discrete DNA nanostructures containing covalent junctions requires reliable methods for the synthesis and assembly of branched oligodeoxynucleotide (ODN) conjugates. This study reports an approach for self-assembly of hard-to-obtain primitive discrete DNA nanostructures“nanoethylenes”, dimers formed by double-stranded oligonucleotides using V-shaped furcate blocks. We scaled up the synthesis of V-shaped oligonucleotide conjugates using pentaerythritol-based diazide and alkyne-modified oligonucleotides using copper­(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and optimized the conditions for “nanoethylene” formation. Next, we designed nanoethylene-based “nanomonomers” containing pendant adapters. They demonstrated smooth and high-yield spontaneous conversion into the smallest cyclic product, DNA tetragon aka “nano-methylcyclobutane”. Formation of DNA nanostructures was confirmed using native polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) and additionally studied by molecular modeling. The proposed facile approach to discrete DNA nanostructures using precise adapter-directed association expands the toolkit for the realm of DNA origami.

Pairing nanoarchitectonics of oligodeoxyribonucleotides with complex diversity: concatemers and self-limited complexes

“… 1–20 Bimolecular V-shape complexes and complexes of other molecularity can be created by means of various non-nucleotide linkers of different lengths, rigidity levels, charges, and hydrophobicity values. 21,23 Moreover, potentially, triplex formation by linkers, non-canonical base pairing, duplex blocks' interactions (for example, similar to those reported for DNA crossovers 61 ), introduction of a chemical modification into oligonucleotides, the formation of catenanes, 62 and other DNA structural factors could affect the efficiency of the self-association of DNA oligomers' and complexes' types formed at thermodynamic equilibrium. In addition, cations significantly stabilize self-limited complexes by shielding electrostatic repulsion and/or via coordination of phosphate residues.…”

“…Self-limited complexes of various molecularity levels can be obtained by varying duplex block length, linker length, and nucleotide content and terminal nucleotides in the duplex blocks and by bending the duplex block. 21,22,28,33,34 Efficient assembly of a self-limited complex can be realized if lengths of the blocks are the same. For example, we found that oligonucleotides with duplex-forming blocks of 20 nt can give rise to self-limited complexes without any linkers (Fig.…”

“…1a–f). 21–23 Native oligonucleotides ( M and N ) without linkers (L m or L n ) form long linear concatemer complexes (Fig. 1a and b).…”

“…Various chemical modifications have been devised to build complexes of predetermined sizes and shapes. 21,22,28,33,34 It has been demonstrated that DNA complexes free of chemical modifications and containing oligothymidylate linkers between complementary blocks can assemble into self-limited complexes. 22 On the one hand, such native complexes of one, two, or more oligonucleotides can be used for building nanoconstructs; on the other hand, an unsuitable type of complex can arise, for example, via hybridization chain reaction.…”

“…1c). 21,23 The same principle is usually applied to tetra-, hexa-, and higher-order complexes. Molecularity of higher-order structures can be determined by enzymatic ligation/digestion assays.…”