Directed assembly of single-stranded DNA fragments for data storage via protein-free catalytic splint ligation.

Oligonucleotides or gene fragments can be ligated in a specified order to create longer DNA assemblies. We present a method where DNA symbols, or oligos designed to encode information for archival data storage, are joined to linker sequences at either end. These linkers dictate the assembly order of the symbols; the order of the symbols can be changed by changing the sequences of the linkers attached to them. Utilizing a ligating DNAzyme as a catalytic splint, we achieve room-temperature, protein-free assembly, offering a cost-effective alternative to traditional enzyme-based ligation methods. We demonstrate this technique by assembling three different five-symbol constructs, with the order of the symbols determined by their linking ends. This linker directed assembly technique allows data-encoding symbols to be assembled in any desired order. Furthermore, the DNAzyme-based assembly method is versatile and can be applied to various DNA assembly applications, particularly where cost-effectiveness and efficient room-temperature ligation are required.