What DNA PolymeraseIs Preferable in miRNA Assay Coupledwith Isothermal Circular Strand Displacement Polymerization Reaction(ICSDPR)?

The products generated in the isothermal circular strand displacement polymerization reaction (ICSDPR) initiated with miRNA-141 were studied. The obtained results demonstrated that if ICSDPR was catalyzed with Large Klenow Fragment (lKF), the canonical duplex and some byproducts, which were not described previously, were observed. The HMW byproducts were shown to be produced as a result of lKF-insisted polymerization of capture hairpin (HP) used in ICSDPR. Contrary to original HP, HMW byproducts are not capture probes because upon polymerization they lack the ability to bind the target. Interestingly, the replacement of lKF with Klenow Fragment (3′-5′ exo-) (KFexo-) prevented the generation of HMW byproducts but did not affect the synthesis of other ICSDPR products. In the presence of both DNA polymerases, the second byproduct, named target-dependent byproduct (TD byproduct), was formed when the capture HP and target sequence formed a perfect duplex. Using an imperfect complex with unpaired nucleotides at the 3′-end of the target sequence prevented the formation of TD byproduct in ICSDPR. The knowledge of mechanisms of the formation of the byproducts and use of KFexo- in catalysis of ICSDPR allowed to develop a highly sensitive plate-based assay of miRNA-141 with the detection limit and sensitivity coefficient of 1.7 fM and 1,400,000 RLU/M, respectively. The amplification index characteristic of KFexo- catalyzed ICSDPR was 22,000. The proposed assay of miRNA-141 showed high specificity toward the target.