Simple in vitro single-stranded linear and circular DNA preparation, functional selection, and validation using phosphor-derived modifications

Interest in the preparation of circular ssDNA library has been increasing recently; therefore, developing a simple and an efficient method for circular DNA generation will be very useful for all procedures and techniques that are dependent on circular ssDNA preparation. In this study, a new simple method for in vitro preparation of circular ssDNA is proposed. We hypothesized that using a phosphorylated-phosphorothioated primer would not affect the efficiency of PCRs but, more importantly, would suppress the activity of the lambda exonuclease enzyme even if it is phosphorylated. The produced phosphorylated ssDNA is ready to be circularized via a ligation reaction using a bridging oligonucleotide. Several optimizations and enhancements have been conducted in the ligation reaction, notably by embedding an extra thymine nucleotide at the ligation site to compensate for the additional adenosine nucleotide added by Taq during the PCR. In addition, the performance of the proposed method has been validated by selecting linear and circular aptamers against Middle East respiratory syndrome coronavirus spike protein during 15 successive cycles of SELEX. Because this new method is simple and user friendly, it has a potential to be automated for high-throughput purposes and may further stir growing interests in preparation of circular ssDNA and its applications.