Simple in-vitro single stranded linear and circular DNA preparation and validation via SELEX using phosphor-derived modifications

Abstract

AbstractInterest in preparation of single stranded circular DNA library has been increasing recently, therefore developing a simple and efficient method for circular DNA generation will be very useful for all procedures and techniques that are dependent on single stranded circular DNA preparation. In this study a new simple method for in vitro preparation of circular single stranded DNA is proposed. We hypothesized that using a phosphorylated-phosphorothioated primer would not affect the efficiency of PCR reactions, but, more importantly, would suppress the activity of Lambda Exonuclease enzyme even if it is phosphorylated. The produced phosphorylated single stranded DNA 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 reaction. In addition, the performance of the proposed method has been validated by selecting linear and circular aptamers against MERS-CoV 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 single stranded circular DNA and its applications.