The programmable construction of synthetic nucleic acid topologies at the molecular level represents a great challenge in chemistry. Achievement of this goal requires the manipulation of the specific Watson-crick base pairing interactions with high precision. Here, we demonstrate that by using the inherent and natural topological properties of nucleic acids and DNA-four-way junctions, which comprise two helical strands to form a node, single-stranded DNA and RNA (ssDNA, ssRNA) knots and links can be created. Using this strategy, we assembled a series of single-stranded nucleic acid topological structures. By precisely designing the curvature and torsion, double-stranded DNA knots can also be realized through a process of annealing complementary strands to the knotted ssDNA template and ligating these strands to seal them in a closed form which preserves the topology of the template knot. Expanding this method, we report two approaches involving either DNA-RNA four-way junctions or DNA-template-directed synthesis of RNA for the construction of new topological structures containing single-stranded RNA free of strong base-pairing interactions. Applying these knots to biological systems as probes, we demonstrated that these structures could effectively function as substrates for DNA topoisomerase and to study the DNA replication under topological constraint. Interestingly, the topological constraint has drastic effects on enzyme activities for both RNA and DNA knots, but the R173A point mutation of <i>E. coli</i> DNA topoisomerase I only abolishes the unknotting activity for ssRNA, but not for ssDNA knots.<br/>

Synthetic nucleic acid topology and their biological applications

254th National Meeting (2017)

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