Directed assembly of single-stranded DNA fragments for data storage via enzyme-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 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, enzyme-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.