Characterization of an ssDNA ligase and its application in aptamer circularization

Abstract

Aptamers are widely used in various biomedical areas as novel molecular recognition elements, however, short single-stranded DNA (ssDNA) or RNA oligonucleotides are easily degraded by nucleases in biological fluids. This problem can be solved by circularizing aptamers with circular ligases. Herein, a moderately thermostable ssDNA ligase was expressed and purified. The purified ligase showed good circularization activity for different length substrates and much higher circularization efficiency than T4 RNA ligase 1. Biochemical characterization revealed that the enzyme showed optimal circularization activity at pH 7.5 and 50 ^ᵒC. Mn²⁺ and Mg²⁺ increased enzyme circularization activity, with Mn²⁺ having higher activity than Mg²⁺. The optimal concentrations of Mn²⁺ and ligase were 1.25-2.5 mM and 0.02 nM, respectively. The kinetic parameters K_m, V_max and K_cat of ssDNA ligase were 1.16 μM, 10.71 μM/min, and 10.7 min^-1, respectively. The ssDNA ligase efficiency was nucleotide-dependent, and 5'-G and 3'-T were the most ligase-favored terminal nucleotides. In addition, the affinity and stability of the circular aptamer were determined. The affinity constant (K_D) was 4.9 μM, and the stability increased compared to its linear form. Molecular docking results showed that the circular aptamer bound to the target via two hydrogen bonds. This study provides a simple and efficient aptamer circularization modification method for improving aptamer stability and expanding its applications.

Keywords: Circular aptamer; Expression; Ligation reaction; Thermostable ligase; ssDNA ligase.