The
mismatched catalytic hairpin assembly (mCHA), a programmable
oligonucleotide circuit, is one of the promising isothermal amplification
methods used in nucleic acid detection. Its limitations are related
to a high background noise observed due to the target-independent
hybridization of the reacting hairpins (HPs). In this work, it was
shown that the introduction of salts such as NaCl and MgCl2 to HP1/HP2 annealing solutions sharply reduces the background in
mCHA and simultaneously increases the signal-to-background (S/B) ratio.
A comparison of the salts demonstrated the higher activity of MgCl2 as compared to NaCl. A similar effect of reducing the background
was observed with a decrease in the concentration of H1/H2 probes
in annealing solutions. Using the favorable annealing conditions allowed
the development of an ultrasensitive chemiluminescence assay coupled
with mCHA for miRNA quantitation. Except mCHA, the use of a streptavidin–polyHRP
conjugate and an enhanced chemiluminescence reaction additionally
increased the assay sensitivity. Notably, the optimization of the
HP annealing diminished the detection limit of the assay by 2 orders
of magnitude and increased the sensitivity and precision of miRNA-141
determination. The discovered fact of reducing the background by the
variation of HP annealing conditions may be valuable not only for
the mCHA performance but also likely for other HP-based biochemical
methods.
In the present work, we describe the development of a chemiluminescent enzyme-linked oligonucleotide assay coupled with mismatched catalytic hairpin assembly (mCHA) amplification for the quantitative determination of microRNA-155. To improve its sensitivity, a polymerase-free mCHA reaction was applied as an isothermal amplification method. The detection limit of the proposed assay was 400 fM. In addition, the high specificity of the assay was demonstrated. The proposed assay allowed assessment of the content of microRNA-155 in human cancer lines such as HepG2, Caco2, MCF7, and HeLa. The quantitation of microRNA-155 was performed after purification of short RNAs (less than 200 nt) from cell lysates since a high matrix effect was observed without this pre-treatment. The results of the quantitative determination of the microRNA content in cells were normalized using nematode microRNA-39, the concentration of which was determined using a heterogeneous assay developed by us using a strategy identical to that of the microRNA-155 assay.
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