Characterization of an ssDNA ligase and its application in aptamer circularization

• Published in: Analytical biochemistry 2024-01, Vol.685, p.115409-115409, Article 115409
• Main Authors: Ma, Zhenxia, Chen, Han, Yang, Yao, Gao, Siyi, Yang, Jiaping, Cui, Shihai, Zhou, Shiyuan, Jiang, Boyang, Zou, Bin, Sun, Mingjuan, Wang, Lianghua
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - chemistry; DNA, Single-Stranded; Ligases - metabolism; Molecular Docking Simulation; RNA - chemistry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2023.115409

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 C. Mn and Mg increased enzyme circularization activity, with Mn having higher activity than Mg . The optimal concentrations of Mn and ligase were 1.25-2.5 mM and 0.02 nM, respectively. The kinetic parameters K , V and K of ssDNA ligase were 1.16 μM, 10.71 μM/min, and 10.7 min , 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 ) 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.