Abstract
DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly. Drawing inspiration from solid-phase synthesis, here we describe a DNA assembly method that uses time, or order of addition, as a parameter to define structural complexity. DNA building blocks are sequentially added with in-situ ligation, then enzymatic enrichment and isolation. This yields a monodisperse, single-stranded long product (for example, 1,000 bases) with user-defined length and sequence pattern. The building blocks can be repeated with different order of addition, giving different DNA patterns. We organize DNA nanostructures and quantum dots using these backbones. Generally, only a small portion of a DNA structure needs to be addressable, while the rest is purely structural. Scaffolds with specifically placed unique sites in a repeating motif greatly minimize the number of components used, while maintaining addressability. This combination of symmetry and site-specific asymmetry within a DNA strand is easily accomplished with our method.
Highlights
DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly
From customized genes1 to high-density data storage tools2 to backbones for structurally diverse nanostructures3–6, there is a tremendous interest in developing synthetic protocols for the production of sequence-defined DNA that is longer than what conventional solid phase synthesis can achieve (4200 bp)
Current methods to produce long, patterned DNA fall into three main categories: stepwise or parallel ligations, usually with intervening purification12–14 or enzymatic selection15, overlap extension or related polymerase chain reaction (PCR) techniques, using many short overlapping fragments1,16,17, and enzymatic amplifications like rolling circle amplification (RCA)7,18
Summary
DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly. The entire process can be done in a single day, using basic molecular biology instrumentation Using this strategy, we generated monodisperse products with a range of sizes (480– 1,058 bases) and sequence patterns, and demonstrated their ability to serve as addressable backbones for building DNA nanostructures or organizing cargo. The oligonucleotides themselves are synthesized using time and order of addition, to grow complex sequences from four simple building blocks (automated solidphase phosphoramidite synthesis) This mechanism allows a user to generate any arbitrary oligonucleotide sequence rapidly and from the same simple starting materials, and is responsible for the widespread use of synthetic DNA throughout the biological sciences. This technique could open the door to accessible ‘designer’ DNA templates for the rapid generation of complex libraries of DNA nanostructures in a practical, cost-efficient manner
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