Mitigating the non-specific rolling circle amplification: a comprehensive assessment of the role of ligation and digestion methods

Rolling circle amplification (RCA) has been utilized for detecting a diverse range of analytes, molecular pathways, and in cellular imaging. However, non-specific amplification (NSA, amplification in absence of a target analyte) in RCA assays, especially those involving pre-synthesized circular DNA (cDNA), affects its sensitivity and specificity. NSA could originate from inefficient ligation or the succeeding cDNA purification steps. To quantify the NSA in RCA here, cDNA substrates were prepared using either self-annealing, splint-padlock, or cohesive end ligations. The cDNAs were then each subjected to nine different exonuclease digestion steps and quantified for NSA under both linear and hyperbranched RCA conditions. We investigated buffer composition, divalent ion concentration, single or dual enzyme digestion, overhang length in cohesive end ligation, and splint length in splint-padlock ligation. When applied in tandem, the conditions successfully mitigated the NSA end-fluorescence 30 – 100 fold while reducing the relative NSA (with respect to primer assisted RCA) to ~5%. Besides understanding the mechanistic origin of NSA, novel aspects of enzyme-substrate selectivity, buffer composition, and the role of divalent ions in enzyme activity were discovered. With increasing analytical applications, this study will help standardize NSA-free RCA assays involving pre-synthesized cDNA.