DNA minicircles with gaps for versatile functionalization.

The programmable self-association of molecular units into higher ordered structures plays a key role in the bottom-up construction of nanomaterials. Crucial for the successful supramolecular assembly of nanoobjects, however, is the choice of the functional molecular units themselves. Nucleic acids have emerged as a convenient target for these purposes because of their unique properties in molecular recognition and the ease by which oligonucleotides can be accessed synthetically. The assembly of DNA nanoobjects with defined twoor three-dimensional geometries requires either rigid subunits, such as in double crossover (DX) or paranemic crossover (PX) elements, or inherently rigid triangular shapes such as tetrahedra or bipyramids. From a structural point of view, DNA minicircles are probably the simplest rigid objects with a nanometer size. Small DNA circles were first prepared by designing two 21mer DNA precursor sequences which, upon hybridization and ligation, resulted in a statistical distribution of DNA minicircles containing 105, 126, 147, and 168 base pairs (bp). Atomic force microscopy (AFM) analysis of 168-bp minicircles confirmed their smooth circular structure without any ring deformation or supercoiling. These features predestine them as building blocks for the assembly of objects on the nanometer scale. However, a major drawback of DNA minicircles in the construction of higher ordered DNA architectures is their unbranched, continuously doublestranded (ds) nature that prevents the guided aggregation of multiple rings. Furthermore, the statistical assembly of the short oligonucleotides that were applied in the known strategies for the synthesis of DNA minicircles prevents the controlled introduction of “customized” sequences into the circle that can serve as defined handles for the selfassembly of multiple rings. We recently reported the assembly of two DNA minicircles, guided by a “strut” of Dervan-type polyamides that specifically held two rings together by binding to different 9mer double-stranded “custom” sequences that were present in each 168-mer ring exactly once. Here we report on the construction of minicircles that contain a customized singlestranded gap sequence at a defined position. The gap can serve as a versatile site for the modification of DNA minicircles, thereby allowing, for example, their guided modification with functional groups through hybridization with synthetic oligonucleotides. The key step in the preparation of DNA minicircles with sequence-specific functionalization focuses on a preformed incomplete minicircle MCgap containing a 21-nucleotide single-stranded (ss) gap region (Figure 1). Hybridizing and