Synthesis and “DNA Interlocks” Formation of Small Circular Oligodeoxynucleotides

Circular oligodeoxynucleotides (c-ODNs) have their particular characteristics in topological properties. However, different from oligoribonucleotides, enzymatic synthesis of small c-ODNs is still challenging using conventional methods. Herein, we successfully achieved highly efficient cyclization of linear single-stranded ODNs using T4 DNA ligase simply through the frozen/lyophilization/cyclization (FLC) method. We successfully shortened the cyclization length of linear ODNs to 20 nt (l-ODN 20) with up to 63% yield, which was never achieved before through normal enzymatic methods. With the efficient FLC method, we further developed "DNA interlocks" which were intercross-linked with multiple c-ODNs using the one-pot FLC method. This FLC strategy provides a powerful, cheap, and convenient method to synthesize small c-ODNs for studying DNA nanotechnology and paves the way to achieve future deciphering of c-ODN functions.