Optimizing the expression and characterization of a thermostable ssDNA ligase from a Thermus scotoductus bacteriophage TS2126

Abstract

Abstract Although aptamers are widely used in various biomedical areas as novel molecular recognition elements, they are degraded by nucleases in biological fluids due to short single-stranded DNA (ssDNA) or RNA oligonucleotides. Research shows that this problem can be solved by circularizing aptamers with circular ligases, which can catalyze the intramolecular ligation of ssDNA templates with 5'-phosphate and 3'-hydroxyl groups. Herein, a thermostable ssDNA ligase was successfully expressed and purified for subsequent aptamer cyclization. The purified ligase showed good circularization activity for substrates with different lengths and had much higher circularization efficiency than T4 RNA ligase 1. Moreover, circularization reaction conditions, such as the concentration of MnCl2, enzyme and betaine, were studied to ascertain the optimal conditions for the catalytic reaction. The results indicated that the highest ligating activity occurred at MnCl2, enzyme and betaine concentrations of 1.25–2.5 mM, 0.02 mM and 1 М, respectively. Based on these, templates with different terminal nucleotides were tested to assess the sequence dependence of ligase. The results show that the efficiency of circularization is sequence dependent, in which 5'-G and 3'-T are the terminal nucleotides most favored by ligase. Finally, the stability asssy further confirmed that the circular aptamer was more stable than its linear form in biological fluids.