Circular single-stranded DNA is a type of single-stranded DNA (ssDNA) with a covalently closed topology. This structure provides several advantages, including resistance to exonuclease degradation, improved thermodynamic stability, and the ability to be replicated by a DNA polymerase in a rolling circle manner. Due to these advantages, circular ssDNAs are finding increasing applications in molecular cloning, disease therapy and diagnosis, nanobiotechnology, microRNA regulation, and engineering special nanoscale subjects. For instance, circular ssDNAs can be utilized as templates for bioanalytical signal amplification and for the synthesis of nano-/micro-/macro- biomaterials through rolling circle amplification (RCA) and rolling circle transcription (RCT) technologies. Circular ssDNAs have also been used as the scaffolds for the self-assembly of versatile DNA origami. Finally, circular ssDNAs have been implemented as theranostic aptamers, miRNA inhibitors, as well as clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) gene editing donors.

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