Enzyme-free Ligation of Oligonucleotides

What is it about?

In this paper we report a new regioselective strategy to form a 600mer in a simultaneous reaction of six individual oligonucleotides approximately 100 nt in length. We used a DNA nanostructure, similar to DNA origami, consisting of six 100mers and 19 shorter connecting strands. The addition of a carbodiimide lead to the formation of the desired phosphodiester bonds. We could show that the final product can be amplified by polymerase chain reaction. The use of a DNA nanostructure to assemble the individual strands in a tightly packed form, increased the yield of the ligation reaction, limited the reaction time in regard to linear duplexes and suppressed side product formation, which is often caused by cross-hybridization of strands.

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Why is it important?

The efficient ligation of multiple DNA strands to form artificial genes and genomes is still a challenge and becomes increasingly important with the development of new tools for molecular biology and medicine. While modern DNA solid phase synthesis grants good yields and high purity oligonucleotides, it is still limited to a length of approximately 150 nt. In order to form longer strands, todays methods mostly use enzymatic approaches and multi step ligations are necessary. Chemical ligation methods, as a cost efficient alternative to enzymes, are being developed. But they often rely on the modification of individual nucleotides, like, for example in CLICK chemistry. Previous ligation reactions to form phosphodiester bonds utilized carbodiimides and are so far slow and low yielding, which makes mutliple parallel ligations not feasible. This new approach could lead to a cheap alternative to established methods and is not limited by sequence, chemical modifications of oligonucleotides and can be scaled up easily.

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